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Pharmacological Characteristics and Control Efficacy of a Novel SDHI Fungicide Pydiflumetofen Against Sclerotinia sclerotiorum
Abstract
Pydiflumetofen is a novel succinate dehydrogenase inhibitor fungicide. In the current research, we determined the sensitivity of 166 Sclerotinia sclerotiorum strains to pydiflumetofen using mycelial growth inhibition method. The results suggest that pydiflumetofen exhibited a strong inhibitory activity against S. sclerotiorum and the EC50 values ranged from 0.0058 to 0.0953 μg ml-1, with a mean EC50 value of 0.0250 μg ml-1. However, the baseline sensitivity was not normally distributed because of a high variation factor. After treatment with pydiflumetofen, cell membrane permeability increases, but exopolysaccharide and oxalic acid production decreases, which contributes to reduced virulence of S. sclerotiorum and leads to failure of disease infection. In addition, protective and curative activity was performed on detached oilseed rape leaves by artificial inoculation. Pydiflumetofen exhibited excellent protective and curative effects against S. sclerotiorum on oilseed rape, and the protective effect was better than the curative effect. Further, field trials were conducted to evaluate the potential of pydiflumetofen in controlling Sclerotinia stem rot (SSR) caused by S. sclerotiorum on oilseed rape. Compared with the currently used fungicides, pydiflumetofen not only exhibited excellent control efficacy against SSR, but also dramatically reduced the dosage of fungicides in the field. Thus, this study provides important references for revealing pharmacological mechanism of pydiflumetofen against S. sclerotiorum and managing SSR on oilseed rape caused by benzimidazole- and dicarboximide-resistant populations.
... Succinate dehydrogenase inhibitors (SDHIs) have been used to control the cereal disease for several decades due to their excellent inhibition activity to a variety of fungal pathogens. [17][18][19][20] Pydiflumetofen is a novel SDHI of pyrazole amide fungicide developed by Syngenta Crop Protection, 21 and like many other SDHIs, including carboxin, boscalid, isopyrazam, and fluopyram, it could interrupt fungal respiration by inhibiting the activity of SDH. 22,23 Point mutations on SdhB, and SdhC led the fungal pathogens to obtain resistance to pydiflumetofen. ...
... Pydiflumetofen, a novel SDHI fungicide, has been registered for controlling many plant fungal diseases due to the broad spectrum and ultra-high-control efficiency. 17,20,24,25,28 It shows strong antifungal activity against many fungal pathogens, including Didymella bryoniae, S. sclerotiorum, Botrytis cinerea, F. asiaticum and Fusarium graminearum. 17,20,24,25,29,[35][36][37] Pydiflumetofen resistance risks have been characterized in some plant pathogens, and point mutations on the subunits SdhB, SdhC, and SdhD of SDH could result in fungicide resistance in those fungi. ...
... 17,20,24,25,28 It shows strong antifungal activity against many fungal pathogens, including Didymella bryoniae, S. sclerotiorum, Botrytis cinerea, F. asiaticum and Fusarium graminearum. 17,20,24,25,29,[35][36][37] Pydiflumetofen resistance risks have been characterized in some plant pathogens, and point mutations on the subunits SdhB, SdhC, and SdhD of SDH could result in fungicide resistance in those fungi. 17,24,25,37 In the current study, we found that pydiflumetofen has excellent antifungal activity against M. oryzae, and could be used as the potential fungicides to control the rice blast disease. ...
BACKGROUND
Rice (Oryza sativa) is an important cereal crop around the world, and has constantly been threaten by the most destructive fungus Magnaporthe oryzae. Pydiflumetofen, a novel succinate dehydrogenase inhibitor (SDHI), is currently being used for controlling various fungal diseases. However, the potential resistance risk of M. oryzae to pydiflumetofen has remained unclear to date, and finding the resistance mechanism is critical for the usage of this fungicide.
RESULTS
The M. oryzae strain Guy11 is sensitive to pydiflumetofen, with EC50 value of 1.24 μg mL⁻¹. 58 pydiflumetofen‐resistant (PR) mutants were obtained through pydiflumetofen‐induced spontaneous mutation, with a mean EC50 value >500 μg mL ⁻¹, and the resistance factor (RF) >400. The PR mutants displayed positive cross‐resistance to carboxin, but were more sensitive to fluopyram. Sequencing analysis showed that all PR mutants presented a cytosine‐to‐thymine transition at nucleotide position +1218, resulting in a replacement of histidine 245 by tyrosine (H245Y) on MoSdhB. The mutation of MoSdhB exhibited strong resistant phenotype, but no detectable growth deficits in fungal development, including vegetative growth and pathogenicity of M. oryzae. An allele‐specific PCR for rapid detection of the H245Y mutants was established in M. oryzae.
CONCLUSION
The M. oryzae is sensitive to pydiflumetofen, and shows a medium to high resistance risk to pydiflumetofen. A point mutation of MoSdhB (H245Y) is responsible for the fungal resistance to pydiflumetofen in M. oryzae. © 2022 Society of Chemical Industry.
... 17 Pydiflumetofen (Fig. S1) is a novel SDHI chiral fungicide that has high efficiency and broad-spectrum bioactivity. For example, a few studies 18,19 have reported that pydiflumetofen exhibited strong inhibitory activity against Sclerotinia sclerotiorum with a mean EC 50 value of 0.0250 μg mL −1 . Neves et al. 20 established the baseline sensitivity of Cercospora zeae-maydis with a mean EC 50 value of 0.0040 μg mL −1 , and Hou et al. ...
... 2.3 Cell membrane permeability of S. sclerotiorum Previous research has shown that pydiflumetofen could not only inhibit the mycelial growth of S. sclerotiorum, but also impair the membrane structure of hyphae. 18,19 To explore the enantioselective effect of pydiflumetofen enantiomers against the membrane structure of hyphae, the cell membrane permeability of S. sclerotiorum was determined. A previous method with slight modification was used to determine the stereoselective effect of pydiflumetofen on the cell membrane permeability of S. sclerotiorum. ...
... As previous work described, pydiflumetofen could decrease the production of exopolysaccharide and oxalic acid. 18,19 The results of the present study indicate that only one of the enantiomers of currentuse pydiflumetofen (R enantiomer) would decrease the production of the exopolysaccharide and oxalic acid. Oxalic acid secreted by S. sclerotiorum can suppress host defenses by manipulating the host redox environment and can enhance the virulence of S. sclerotiorum in different ways. ...
BACKGROUND
Pydiflumetofen, as a new succinate dehydrogenase inhibitor (SDHI) chiral fungicide, has been used in crop production because of its broad‐spectrum and high‐efficiency antifungal activity. However, little is known about pydiflumetofen at the chiral level. The stereoselective bioactivity and degradation of pydiflumetofen enantiomers were therefore investigated.
RESULTS
Pydiflumetofen presented effective bioactivity against the eight tested phytopathogens, and its enantiomers showed significant differences in activity. The bioactivity of R‐pydiflumetofen was 9.0–958.8 times higher than that of the S enantiomer. Treatment with R‐pydiflumetofen increased the cell membrane permeability of Sclerotinia sclerotiorum and decreased exopolysaccharide and oxalic acid production more than treatment with S‐pydiflumetofen. Furthermore, R‐pydflumetofen exhibited better inhibitory activity against the succinate dehydrogenase enzyme of S. sclerotiorum than S‐pydiflumetofen by 584‐fold. According to homology modeling and molecular docking studies, the binding affinities of the R and S enantiomers were −7.0 and −5.3 kcal mol⁻¹, respectively. Additionally, the degradation half‐lives of S‐ and R‐pydiflumetofen in three vegetables (cucumber, eggplant, and cowpea) under field conditions were 2.56–3.12 days and 2.48–2.76 days, respectively, which reveals that R‐pydiflumetofen degrades faster than S‐pydiflumetofen.
CONCLUSION
Based on the results obtained, R‐pydiflumetofen not only exhibited a higher bactericidal activity, but also posed fewer residual risks in the environment. The mechanism of the stereoselective bioactivity was correlated with the stereoselective inhibition activity of the target enzyme and affected the cell membrane permeability and the production of exopolysaccharide and oxalic acid. This research could provide a foundation for the systematic evaluation of pydiflumetofen from an enantiomeric view. © 2021 Society of Chemical Industry.
... The picoxystrobin sensitivity of N. clavispora was performed with 56 strains, using the mycelial growth inhibition method, as described previously [15][16][17][18]. Mycelial plugs (4 mm diameter) were taken from 5-day-old colonies and transferred onto AEA plates amended with 0, 0.015625, 0.03125, 0.0625, 0.125, 0.25 and 0.5 µg/mL of picoxystrobin each. ...
... N. clavispora strains ZJJ5, ZJJ9 and ZJJ12 were selected to determine the cell membrane permeability in the presence of picoxystrobin, according to previous studies [17,18], with minor modification. A total of 15 mycelial plugs were taken from 5-day-old colonies and transferred into 100 mL liquid AEA media. ...
Vine tea (Ampelopsis grossedentata) is a traditional herb widely consumed in southwestern China that possesses paramount potential for human health. In 2021, the outbreak of typical leaf blight disease was observed in almost all vine tea plantations in Zhangjiajie of Hunan province, resulting in significant economic losses of vine tea production. In this study, we identified Neopestalotiopsis clavispora as the causal agent of vine tea leaf blight via its morphological characteristics and molecular identification. The sensitivity distribution of N. clavispora isolates to picoxystrobin were determined based on mycelial growth and spore germination inhibition assays. The EC50 values for mycelial growth ranged from 0.0062 to 0.0658 µg/mL, with a mean of 0.0282 ± 0.0148 µg/mL. The EC50 values for spore germination ranged from 0.0014 to 0.0099 µg/mL, and the mean value was 0.0048 ± 0.0022 µg/mL. Picoxystrobin increased fungal cell membrane permeability, but inhibited fungal ATP biosynthesis. Moreover, picoxystrobin exhibited good in planta control efficacy on vine tea leaves. Three picoxystrobin-resistant mutants were obtained in the current study, but no mutations were detected in the N. clavispora Cytb gene. Competitive ability assays showed that the conidium production and pathogenicity of all picoxystrobin-resistant mutants decreased as compared to their progenitors, indicating that picoxystrobin-resistant mutants suffer fitness penalty. These findings provide important evidence for picoxystrobin in vine tea leaf blight management and increase understanding of the resistance mechanism of picoxystrobin against N. clavispora.
... Most of the newly developed fungicides are based on the original reactive group as a backbone. At present, SDHIs have been widely applied for controlling many plant diseases [15][16][17][18]. However, SDHIs are rarely used to control FHB, especially in the control of DON production in wheat grains. ...
... Previous studies reported that SDHIs have good effects in the prevention and control of other diseases. For example, pydiflumetofen can effectively inhibit Sclerotinia sclerotiorum, with an average EC 50 value of 0.0250 µg/mL [18]. In addition, boscalid and isopyrazam have a better inhibitory effect on mycelial growth of Aspergillus flavus than Fusarium species. ...
Deoxynivalenol (DON) is a class of mycotoxin produced in cereal crops infected with Fusarium graminearum species complex (FGSC). In China, FGSC mainly includes Fusarium asiaticum and F. graminearum. DON belongs to the trichothecenes and poses a serious threat to the safety and health of humans and animals. Succinate dehydrogenase inhibitors (SDHIs) are a class of fungicides that act on succinate dehydrogenase and inhibit the respiration of pathogenic fungi. In this study, the fungicidal activities of five SDHIs, including fluopyram, flutolanil, boscalid, benzovindiflupyr, and fluxapyroxad, against FGSC were determined based on mycelial growth and spore germination inhibition methods. The five SDHIs exhibited better inhibitory activities in spore germination than mycelial growth. Fluopyram exhibited a higher inhibitory effect in mycelial growth and spore germination in comparison to the other four SDHIs. In addition, the biological characteristics of F. asiaticum as affected by the five SDHIs were determined. We found that these five SDHIs decreased DON, pyruvic acid and acetyl-CoA production, isocitrate dehydrogenase mitochondrial (ICDHm) and SDH activities, and NADH and ATP content of F. asiaticum but increased the citric acid content. In addition, TRI5 gene expression was inhibited, and the formation of toxisomes was disrupted by the five SDHIs, further confirming that SDHIs can decrease DON biosynthesis of F. asiaticum. Thus, we concluded that SDHIs may decrease DON biosynthesis of F. asiaticum by inhibiting glycolysis and the tricarboxylic acid (TCA) cycle. Overall, the findings from the study will provide important references for managing Fusarium head blight (FHB) caused by FGSC and reducing DON contamination in F. asiaticum-infected wheat grains.
... There are no cultivars with high resistance that can be used in production (Zhao et al., 2009). Although new fungicides are continually being developed to prevent Sclerotinia diseases, the frequency of fungicide-resistant strains of S. sclerotiorum in the field is increasing (Duan et al., 2018(Duan et al., , 2019Tao et al., 2021). Sclerotinia disease is highly challenging to control and causes huge economic losses annually. ...
Many plant pathogens secrete effector proteins into the host plant to suppress host immunity and facilitate pathogen colonization. The necrotrophic pathogen Sclerotinia sclerotiorum causes severe plant diseases and results in enormous economic losses, in which secreted proteins play a crucial role. SsCVNH was previously reported as a secreted protein, and its expression is significantly upregulated at 3 h after inoculation on the host plant. Here, we further demonstrated that deletion of SsCVNH leads to attenuated virulence. Heterologous expression of SsCVNH in Arabidopsis enhanced pathogen infection, inhibited the host PAMP‐triggered immunity (PTI) response and increased plant susceptibility to S. sclerotiorum . SsCVNH interacted with class III peroxidase AtPRX71, a positive regulator of innate immunity against plant pathogens. SsCVNH could also interact with other class III peroxidases, thus reducing peroxidase activity and suppressing plant immunity. Our results reveal a new infection strategy employed by S. sclerotiorum in which the fungus suppresses the function of class III peroxidases, the major component of PTI to promote its own infection.
... The heterocyclic fragment is connected to the carbonyl group, and the N atom at the other end is usually connected to a hydrophobic group [11]. Pydiflumetofen, an efficient and broadspectrum SDHI fungicide, was marketed by Syngenta in 2017 and has been registered for use in potatoes, soybean, oilseed rape, grapes, and peanuts [12]. However, the risk of Pydiflumetofen resistance develops quickly in Fusarium asiaticum [13]. ...
Succinate dehydrogenase (SDH, EC 1.3.5.1) is one of the most promising targets for fungicide development and has attracted great attention worldwide. However, existing commercial fungicides targeting SDH have led to the increasingly prominent problem of pathogen resistance, so it is necessary to develop new fungicides. Herein, we used a structure-based molecular design strategy to design and synthesize a series of novel SDHI fungicides containing an N-(alkoxy)diphenyl ether carboxamide skeleton. The mycelial growth inhibition experiment showed that compound M15 exhibited a very good control effect against four plant pathogens, with inhibition rates of more than 60% at a dose of 50 μg/mL. A structure–activity relationship study found that N-O-benzyl-substituted derivatives showed better antifungal activity than others, especially the introduction of a halogen on the benzyl. Furthermore, the molecular docking results suggested that π–π interactions with Trp35 and hydrogen bonds with Tyr33 and Trp173 were crucial interaction sites when inhibitors bound to SDH. Morphological observation of mycelium revealed that M15 could inhibit the growth of mycelia. Moreover, in vivo and in vitro tests showed that M15 not only inhibited the enzyme activity of SDH but also effectively protected rice from damage due to R. solani infection, with a result close to that of the control at a concentration of 200 μg/mL. Thus, the N-(alkoxy)diphenyl ether carboxamide skeleton is a new starting point for the discovery of new SDH inhibitors and is worthy of further investigation.
... It inhibits the mitochondrial function by interfering with the tricarboxylic acid (TCA) cycle on the respiratory electron transfer chain complex II, which consequently hampers energy production, inhibits pathogen growth, and ultimately leads to its death (see chemical structural formula in Figure 1) [18,19]. This is an efficient and broad-spectrum agent, suitable for controlling various diseases in various crops, with good effects on wheat scab, soybean leaf spot, potato early blight, etc. [20]. Currently, global research on pydiflumetofen has focused on its efficacy, prevention and control, toxicology, and residual analysis methods [21,22]. ...
Pydiflumetofen is a potent fungicide that effectively inhibits pathogenic fungal growth by regulating succinate dehydrogenase activity. It effectively prevents and treats various fungal diseases, including leaf spot, powdery mildew, grey mold, bakanae, scab, and sheath blight. Pydiflumetofen’s hydrolytic and degradation properties were investigated indoors in four distinct soil types (phaeozems, lixisols, ferrosols, and plinthosols) to assess its risks in aquatic and soil environments. The effect of soil physicochemical properties and external environmental conditions on its degradation was also explored. Hydrolysis experiments found that pydiflumetofen’s hydrolysis rate decreased with increasing concentration, regardless of the initial concentration. Furthermore, an increasing temperature significantly enhances the hydrolysis rate, with neutral conditions having higher degradation rates than acidic and alkaline conditions. Pydiflumetofen showed a degradation half-life of 10.79–24.82 days and a degradation rate of 0.0276–0.0642 in different soils. Phaeozems soils had the fastest degradation, while ferrosols soils had the slowest. Sterilization significantly reduced its soil degradation rate and extended its half-life, which confirmed that microorganisms were the primary cause. Therefore, when using pydiflumetofen in agricultural production activities, the characteristics of water bodies, soil, and environmental factors must be considered, while minimizing the emissions and environmental impact.
... The succinate dehydrogenase inhibitor (SDHI) group of fungicides is highly effective against a broad range of fungi and widely used to control many plant diseases [10,[15][16][17]. However, since SDHI fungicides have a single target site, many plant pathogens have already developed resistance. ...
Fusarium head blight (FHB), which is primarily caused by Fusarium graminearum, is a widespread and devastating disease of wheat. In the absence of resistant varieties, the control of FHB relies heavily on the application of fungicides, and the new generation SDHI fungicide, pydiflumetofen, has recently been registered in China for the control of FHB in wheat. The current study explored three genetically stable, highly resistant laboratory mutants (S2-4-2R, S27-3R, and S28-2R, with EC50 values of 25.10, 28.57, and 19.22 μg/mL, respectively) to investigate the potential risks associated with pydiflumetofen resistance. Although the mycelial growth of the mutants differed little compared to their parental isolates, the study found that the resistant mutants exhibited significantly reduced (p < 0.05) levels of sporulation and pathogenicity, which suggests a significant fitness cost associated with pydiflumetofen resistance in F. graminearum. Sequence analysis of the Sdh target protein identified numerous amino acid substitutions in the predicted sequences of the four subunits: FgSdhA, FgSdhB, FgSdhC, and FgSdhD. Indeed, the mutants were found to have a series of substitution in multiple subunits such that all three exhibited five identical changes, including Y182F in the FgSdhA subunit; H53Q, C90S, and A94V in FgSdhB; and S31F in FgSdhC. In addition, gene expression analysis revealed that all of the FgSdh genes had significantly altered expression (p < 0.05), particularly FgSdhA and FgdhC, which exhibited remarkably low levels of expression. However, the study found no evidence of cross-resistance between pydiflumetofen and tebuconazole, fludioxonil, prochloraz, fluazinam, carbendazim, pyraclostrobin, or difenoconazole, which indicates that these fungicides, either in rotation or combination with pydiflumetofen, could mitigate the risk of resistance emerging and provide ongoing control of FHB to ensure high and stable wheat yields.
... As a third-generation SDHI fungicide and a broad-spectrum bactericide, pydiflumetofen shows high efficacy in the prevention and control of nuclear disc bacterial and fungal diseases in soybean, wheat, and a variety of vegetables. Pydiflumetofen has substantial advantages in the control of Fusarium, and can be used for the prevention and treatment of soybean brown spot disease, fusarium head blight of wheat, rape sclerotia, and other diseases [9,10]. Pydiflumetofen was first registered in Argentina in 2017, and is one of the most promising pesticides for SDHI [11]. ...
In this study, the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method, combined with high-performance liquid chromatography–tandem mass spectrometry, was chosen for detecting pydiflumetofen residues in soybean plants, soybeans and soil, and assessing the risk of short- and long-term dietary intake. Pydiflumetofen concentrations ranging from 0.001–0.5 mg/L exhibited good linearity (r > 0.997). At varying doses, the average pydiflumetofen recovery rates and relative standard deviations among soybean plants, soybeans, and soil ranged from 83.9 ± 1.1% to 99.5 ± 3.3% and from 0.77 to 7.77%, respectively. The sensitivity, accuracy, and precision of the chosen methodology met the requirements of pesticide residue analysis. The results of the degradation dynamics test showed that the half-life of pydiflumetofen (t1/2) in soybean plants and in soil were 3.6 to 5.7 and from 7.9 to 25.7 d, respectively. Assessment of the concentration of pydiflumetofen residues in soybeans revealed acute and chronic dietary exposure risks of 0.06 and 7.54%, respectively. As these values are very low, pydiflumetofen residues in soybeans present an acceptable risk to public health. The results of this study will help to guide the practical application of pydiflumetofen and minimize the environmental risks associated with its use.
... After incubation for 4 days at 25°C in the dark, the colony diameter was measured, the EC 50 values (50% effective concentration) were calculated by the linear regression of the prohibition of percentage growth inhibition against the log-transformed fungicide concentration. 21 Three replicates were performed for each concentration of each strain. ...
BACKGROUND
Cucumber fruit rot (CFR) caused by Fusarium incarnatum is a devastating fungal disease in cucumber. In recent years, CFR has occurred frequently, resulting in serious yield and quality losses in China. Phenamacril exhibits a specific antifungal activity against Fusarium species. However, no data for phenamacril against F. incarnatum is available.
RESULTS
The sensitivity of 80 F. incarnatum strains to phenamacril was determined. The half maximal effective concentration (EC50) values ranged from 0.1134 to 0.3261 μg mL⁻¹ with a mean EC50 value of 0.2170 ± 0.0496 μg mL⁻¹. A total of seven resistant mutants were obtained from 450 mycelial plugs by phenamacril‐taming on potato dextrose agar (PDA) plates with 10 μg mL⁻¹ of phenamacril, and the resistant frequency was 1.56%. Phenamacril‐resistant mutants showed decreased mycelial growth, conidiation and virulence as compared with the corresponding wild‐type strains, indicating that phenamacril resistance suffered a fitness penalty in F. incarnatum. In addition, using sequence analysis, the point mutations of S217P or I424S were discovered in Fimyosin‐5 (the target of phenamacril). The site‐directed mutagenesis of the S217P, P217S, I424S and S424I substitutions were constructed to reveal the relationship between the point mutations and phenamacril resistance. The results strongly demonstrated that the mutations of S217P and I424S in Fimyosin‐5 conferred phenamacril‐resistance in F. incarnatum.
CONCLUSION
Phenamacril‐resistant mutants were easily induced and their resistance level was high. The S217P or I424S substitutions in Fimyosin‐5 conferring phenamacril resistance were detected and futherly verified by transformation assay with site‐directed mutagenesis. Thus, we proposed that the resistance development of F. incarnatum to phenamacril is high risk. © 2022 Society of Chemical Industry.
... Electrolyte leakages of TR4 induced by Pro enantiomers were measured according to a previously described method with minor modifications [48]. Fresh mycelia (0.5 g) were harvested from PDB after a 7-day incubation and washed twice with sterile deionized water and then suspended in 20 mL sterile deionized water containing different concentrations of Pro enantiomers (0, 0.4 and 0.8 µg/mL). ...
As a typical triazole fungicide, prothioconazole (Pro) has been used extensively due to its broad spectrum and high efficiency. However, as a racemic mixture of two enantiomers (R-Pro and S-Pro), the enantiomer-specific outcomes on the bioactivity have not been fully elucidated. Here, we investigate how chirality affects the activity and mechanism of action of Pro enantiomers on Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), the notorious virulent strain causing Fusarium wilt of banana (FWB). The Pro enantiomers were evaluated in vivo and in vitro with the aid of three bioassay methods for their fungicidal activities against TR4 and the results suggested that the fungicidal activities of Pro enantiomers are stereoselective in a dose-dependent manner with R-Pro making a major contribution to the treatment outcomes. We found that R-Pro led to more severe morphological changes and impairment in membrane integrity than S-Pro. R-Pro also led to the increase of more MDA contents and the reduction of more SOD and CAT activities compared with the control and S-Pro groups. Furthermore, the expression of Cytochrome P450 14α-sterol demethylases (CYP51), the target for triazole fungicides, was significantly increased upon treatment with R-Pro rather than S-Pro, at both transcriptional and translational levels; so were the activities of the Cytochrome P450 enzymes. In addition, surface plasmon resonance (SPR) and molecular docking illuminated the stereoselective interactions between the Pro enantiomers and CYP51 of TR4 at the target site, and R-Pro showed a better binding affinity with CYP51 than S-Pro. These results suggested an enantioselective mechanism of Pro against TR4, which may rely on the enantioselective damages to the fungal cell membrane and the enantiospecific CYP51 binding affinity. Taken together, our study shed some light on the mechanisms underlying the differential activities of the Pro enantiomers against TR4 and demonstrated that Pro can be used as a potential candidate in the treatment of FWB.
... It exhibits considerably high activity against a wide range of fungi in vitro. [21][22][23][24] A total of 18 products containing pydiflumetofen in combination with difenoconazole are approved for use in soybean, peanuts, tomatoes, peppers, and grapes in Argentina. This fungicide is currently being registered in China to control gray mold in a variety of crops. ...
BACKGROUND
Gray mold caused by Botrytis cinerea Pers. is one of the most significant airborne diseases. It can infest a wide range of crops, causing significant losses in yield and quality worldwide. Pydiflumetofen, a new generation succinate dehydrogenase inhibitor (SDHI), is currently being registered in China to control gray mold in a variety of crops. The baseline sensitivity, resistance risk, and resistance mechanism of Botrytis cinerea to pydiflumetofen were assessed in this study.
RESULTS
A total of 138 strains of B. cinerea from 10 different regions were tested for their sensitivity to pydiflumetofen, and the mean EC50 value was 0.0056 μg mL⁻¹. Eight mutants were obtained by fungicide adaption from five sensitive parental isolates, and the resistance factor (RF) ranged from 51 to 135. The mutants exhibited strong adaptive traits in conidial production, conidial germination, and pathogenicity. Positive cross‐resistance was only observed between other SDHIs (i.e. boscalid, fluopyram, and isopyrazam). Two different types of pydiflumetofen‐resistant mutants were identified: point mutation P225L in sdhB and double mutation G85A and I93V in sdhC. The in vivo control efficacy of pydiflumetofen on the resistant mutants carrying P225L in sdhB as well as G85A and I93V in sdhC was significantly decreased to 52.62% and 32.27%, respectively.
CONCLUSION
The fitness was significantly higher for all pydiflumetofen‐resistant mutants than the corresponding parental. Two types of point mutations, sdhB‐P225L and sdhC‐G85A and I93V, might confer resistance to pydiflumetofen in B. cinerea. A precautionary resistance management strategy should be implemented. © 2021 Society of Chemical Industry
... Isolates of S. sclerotiorum from Anhui and Henan Province, China, showed developed resistance against carbendazim, a benzimidazole group of fungicides that binds to beta-tubulin (Xu et al. 2015b;Liu et al. 2018). Therefore, recent researches are mainly focused on alternative fungicides such as pyrisoxazole (Duan et al. 2018), pydiflumetofen (Duan et al. 2019), 3-(2-pyridyl)methyl-2-(4chlorphenyl) iminothiazolidine (PMAS) (Zhang et al. 2020a) or natural fungicides such as Fungastop™ (active ingredient essential oil of mint) and Bion (active ingredient acibenzolar-s-methyl) (Ojaghian et al. 2020) with different modes of action to prevent or delay the onset of the development of fungicide resistance (Table 1). Other than the development of resistance, the other concern is the overuse of chemical fungicides by farmers, which may cause a negative effect on soil organisms and aquatic life. ...
Abstract
Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum (Lib.) De Bary, is one of the most devastating diseases of tomato. The control of the pathogen is challenging because of its high genetic variability, broad host range and lack of information on natural resistance in tomato germplasm. Through the availability of the genome sequence of S. sclerotiorum, progress has been made in the identification of genes associated with the early stages of pathogenesis. However, less than 60 genes have been functionally validated. Very little information is available on the function of genes associated with the later stage of pathogenesis and signalling pathways. Researches related to SSR management have addressed agricultural practices and the application of fungicides. Agricultural practices like field sanitisation, soil sterilisation, biofumigation and organic amendments have shown improvement in disease control, while smart crop monitoring using unmanned aerial systems and optical sensors can further aid in early detection. Application of biological fungicides has demonstrated potential in SSR control in tomato, while the application of ultraviolet radiation can further aid in sustainable disease control. Transgenic approaches via the expression of some genes targeting degradation of the important molecule of pathogenesis or overexpression of the tomato defence-related genes have shown promises to enhance resistance. However, the generation of disease-resistant varieties in tomato is still in infancy. This review focuses on the current advancement of S. sclerotiorum-tomato pathosystem research and identifies some gaps in knowledge while providing recommendations for future research towards sustainable control of SSR in tomato.
... For example, pydiflumetofen provided excellent control efficacy against Fusarium head blight in the field and pydiflumetofen had strong inhibition activities against Fusarium asiaticum in vitro (Hou et al. 2017). Meanwhile, pydiflumetofen exhibited a strong inhibitory activity against S. sclerotiorum (Duan et al. 2019). Pydiflumetofen at 300 mg/L effectively controlled gray mold on cucumber leaves (80.9%), and its efficacy was superior to that of boscalid at 400 mg/L (42.7%), the results suggest that pydiflumetofen provides effective control for the management of gray mold but must be used with caution (He et al. 2020a, b). ...
Pydiflumetofen is a new succinate dehydrogenase inhibitor fungicide, and the method for determination of its residues in rice and associated environmental samples has not yet been reported. Here, we optimized, Quick Easy, Cheap, Effective, Rugged, Safe (QuEChERS) method for sample preparation, and used high performance liquid chromatography–tandem mass spectrometry (HPLC–MS) to detect the residual amounts of pydiflumetofen in rice and its environment. The results showed that there was a good linearity over the pydiflumetofen concentration range of 0.01–0.1 mg/L in all matrices (R2 > 0.99). At the spiked levels of 0.01, 0.05, and 0.1 mg/kg, the recovery rates of pydiflumetofen from various matrices were between 84.23 and 105.10 %, with the relative standard deviation of 1.07–9.99 %. The limit of detection (signal-to-noise ratio = 3) of the proposed method for pydiflumetofen was in the range of 1.9–3.5 µg/kg, and the limit of quantification (signal-to-noise ratio = 10) was in the range of 6.3–11.7 µg/kg.
... It offers effective disease control across multiple crops, protecting them against a broad range of yield-and quality-reducing diseases, such as leaf spots, powdery mildew, molds and Fusarium headblight 19,20 as well as, in particular, Sclerotinia stem rot (Fig. 6). 21,22 The synthesis of pydiflumetofen (III) starts from 2,4,6-trichlorobenzoic acid (15), as it is one of the few trichlorinated benzene building blocks which are available on large scale. Esterification via the acid chloride and reduction of the resulting methyl ester to the benzyl alcohol 16 is followed by Swern oxidation to 2,4,6-trichlorobenzaldehyde 17. Henry reaction of this aldehyde with nitroethane delivers the nitrostyrene 18, which is then transformed under conditions of the Nef reaction to the 2,4,6-trichlorobenzyl methyl ketone 19. ...
In this review, the synthesis of 33 agrochemicals that received an international standardization organization (ISO) name between January 2015 and December 2018 is described. The aim is to showcase the broad range and scope of reactions, reagents and intermediates used to discover and produce the latest active ingredients addressing the crop protection industry’s needs.
... 63,64 A large number of fungicides have an inhibitory role by destroying the integrity of the cell membrane. [65][66][67] It can be inferred that eugenol damaged the cell membrane by inhibiting ergosterol synthesis. 68,69 In addition, eugenol increased the MDA content, which indicated that lipid peroxidation had occurred in R. solani, exacerbating damage to the membrane and inhibiting mycelial growth. ...
BACKGROUND
Rice sheath blight caused by Rhizoctonia solani is a devastating disease of rice in China. However, indiscriminate use of chemical fungicides applied to control the disease raise major environmental and food safety issues. Ecofriendly biocontrol alternatives are urgently needed. Eugenol, one of the main ingredients in Syzygium aromaticum, has attracted much attention owing to its antifungal properties. However, its mode of action is still not clear. Herein, the antifungal activity and mode of action of eugenol against R. solani were investigated.
RESULTS
Results confirmed that the mycelia of R. solani treated with eugenol shrank and became dehydrated, the cytoplasmic wall separated, and the vacuoles and mitochondria decreased or dissolved. Moreover, we found that eugenol downregulated expression of C‐4 methyl sterol oxidase, inhibited synthesis of ergosterol, increased membrane permeability and impaired the transportation of amino acids and glucose across the cell membrane. In addition, eugenol decreased the mitochondrial membrane potential and initiated an oxidative stress reaction by increasing reactive oxygen species and malondialdehyde, which together with membrane damage contribute to the antifungal activity of eugenol. Meanwhile, eugenol might inhibit R. solani by affecting oxidative phosphorylation and the tricarboxylic acid cycle (TCA cycle).
CONCLUSION
In view of its multitarget properties against R. solani, eugenol provides an alternative approach to chemical control strategies against rice sheath blight. © 2021 Society of Chemical Industry.
... Pydiflumetofen has been registered in some countries against different diseases, such as some fungal diseases of potato and grapes in New Zealand, and in combination with difenoconazole against some fungal diseases of soybean, peanut, tomato, pepper, and grape in Argentina (Buxton et al. 2016;Harp et al. 2016;Olaya et al. 2016). Recently in China, pydiflumetofen had shown antifungal activities against in vitro growth of Fusarium asiaticum and Sclerotinia sclerotiorum and diseases caused by them, i.e., Fusarium head blight and Sclerotinia stem rot, respectively (Duan et al. 2019;Hou et al. 2017). This new fungicide has not yet been registered against any rice crop disease in China or anywhere in the world. ...
New fungicides are tools to manage fungal diseases and overcome emerging resistance in fugnal pathogens. In this study, a total of 121 isolates of Fusarium fujikuroi, the causal agent of rice bakanae disease (RBD), were collected from various geographical regions of China, and their sensitivity to a novel succinate dehydrogenase inhibitor (SDHI)fungicide ‘pydiflumetofen’ was evaluated. The 50% effective concentration (EC50) value of pydiflumetofen for mycelial growth suppression ranged from 0.0101 to 0.1012 μg/ml and for conidial germination inhibition ranged from 0.0051to 0.1082 μg/ml. Pydiflumetofen treated hyphae showed contortion and increased branching, cell membrane permeability, and glycerol content significantly. The result of electron microscope transmission indicated that pydiflumetofen damaged the mycelial cell wall and the cell membrane, and almost broken up the cells, which increased the intracellular plasma leakage. There was no cross-resistance between pydiflumetofen and the widely used fungicides such as carbendazim, prochloraz, and phenamacril. Pydiflumetofen was found safe to seeds and rice seedlings of four rice cultivars, used up to 400 μg/ml. Seed treatment significantly decreased the rate of diseased plants in the greenhouse as well as in field trials in 2017 and 2018. Pydiflumetofen showed superb results against RBD, when used at 10 or 20 g a.i./100 kg of treated seeds, providing over 90% control efficacy (the highest control efficacy was up to 97%), which was significantly higher than that of 25% phenamacril (SC) at 10g or carbendazim at 100 g. Pydiflumetofen is highly effective against F. fujikuroi growth and sporulation as well as RBD in the field.
... Pydiflumetofen has been registered for use in potatoes, soybean, oilseed rape, grapes and peanuts. 4,23 Owing to its excellent efficacy against FHB, pydiflumetofen has been, since 2019, launched into the market in China to control wheat FHB. To date, however, no data is available regarding the potential pyd-resistance selected in F. asiaticum, or any reports of resistance mechanisms. ...
BACKGROUND
Fusarium asiaticum is one of predominant pathogens of Fusarium head blight (FHB) in China. Pydiflumetofen (Pyd) is a novel succinate dehydrogenase inhibitor (SDHI) which has been commercialized in China for the controlling of wheat FHB since 2019. In the current study, a risk assessment of the pydiflumetofen‐resistance selected in Fusarium asiaticum was investigated.
RESULTS
One PydMR mutant [resistance factor (RF) < 80] and four PydHR mutants (RF > 3000) were generated by fungicide‐taming from 1000 mycelial discs of the wild‐type strain 2021. Nucleotide sequences alignment results of FaSdh from the wild‐type strain and resistant mutants showed that all the mutations were categorized into three genotypes, i.e. FaSdhBH248Y from PydMR mutant, both FaSdhC1A64V and FaSdhC1R67K from PydHR mutants. All the resistant mutants possessed no fitness penalty based on the data of mycelial linear growth, conidiation and virulence. In addition, the FaSdhC1A64V mutants showed positive cross‐resistance between pydiflumetofen and boscalid or thifluzamide, but no cross‐resistance between pydiflumetofen and Y13149 or Y12196, while the FaSdhC1R67K mutants exhibited positive cross‐resistance between pydiflumetofen and boscalid, thifluzamide or Y12196, and no cross‐resistance between pydiflumetofen and Y13149. Furthermore, positive cross‐resistance between the five tested SDHIs was detected in the FaSdhBH248Y mutants.
CONCLUSION
The results suggest a moderate to high resistance risk of F. asiaticum to pydiflumetofen, and provide essential data for monitoring the emergence of resistance and resistance management strategies for pydiflumetofen, which will be useful for scientific application of this fungicide in China.
... Previous studies have widely reported that other types of fungicides, such as SDHIs, sterile biosynthesis inhibitors (SBIs), phenylpyrroles and dicarboximides, markedly affect cell membrane permeability of filamentous fungi (Duan et al., 2013, 2018a, 2019b, Xin et al., 2020, indicating that the increased cell membrane permeability is not specific response to some fungicides. Additionally, phytochemicals are also reported to cause membrane disturbance resulting in the loss of membrane integrity (Wink, 2003). ...
Quinone outside inhibitors (QoIs) are currently extensively used agricultural fungicides. However, the application of QoIs in controlling Fusarium graminearum was rarely reported. No information is available on pharmacological characteristics of QoIs against F. graminearum, as well as their effects on DON biosynthesis. Here, we found that six QoIs exhibited an excellent fungicidal activity against F. graminearum based on mycelial growth and spore germination. ATP production assay further confirmed that QoIs decreased ATP production via inhibiting mitochondrial respiration, which contributes their fungicidal activity. Unfortunately, QoIs can stimulate DON production and up-regulate the expression of Tri5 and Tri6 genes. Additionally, acetyl-CoA, the basic precursor of DON biosynthesis, significantly increased as affected by QoIs, furtherly indicating that QoIs indeed enhance DON biosynthesis. We also found that QoIs can accelerate the formation of toxisomes and enhance the fluorescence signals of Tri-GFP labeled toxisomes, which may be due to the effect of QoIs on toxisome-related endoplasmic reticulum-remodeling. In addition, QoIs could disrupt the homeostasis of mitochondrial dynamics, resulting in the fragmented mitochondria. Finally, the simulated inoculation assay with wheat grains further verified that QoIs can stimulate DON production relative to wheat grain weight, especially relative to mycelial biomass.
... Given the Priaxor results, we hypothesize that fluxapyroxad, the SDHI active ingredient, does not have the curative activity of the DMIs tested. Although some SDHI (Chen et al. 2014;Duan et al. 2019;He et al. 2017;Veloukas and Karaoglanidis 2012) and QoI (Anesiadis et al. 2003) fungicide active ingredients have limited curative activity in some pathosystems, their efficacy is best realized when applied prophylactically (Mueller et al. 2013). It is important to note that in this study, curative activity of the fungicides was only assessed 5 and 3 days after inoculation. ...
Powdery mildew, caused by the obligate fungal pathogen Blumeria graminis, has been increasing in incidence and severity on wheat in the Chesapeake Bay region of the mid-Atlantic United States. Although fungicides are used for in-season management of powdery mildew, no studies to date have assessed the ability of these products to control disease after pathogen arrival/infection (curative activity) and the duration of disease control provided after fungicide application (protective activity) under controlled conditions. Five commercially available fungicide products, including Caramba, Stratego YLD, Priaxor, Prosaro, and Trivapro, were applied at either 3 or 5 days after inoculation with B. graminis spores to assess curative activity. In a separate study, protective activity for these fungicides was assessed by applying fungicides and inoculating with B. graminis spores the same day and 21 and 42 days after fungicide application. All fungicides reduced powdery mildew severity on foliage compared with nontreated controls. Priaxor provided 11 to 18% less activity than other fungicides when applied curatively. All products provided protective control up to 42 days after inoculation, with Stratego YLD and Priaxor providing the greatest level of control at 68 and 56%, respectively. Our data indicate that different fungicides can have subtle differences in overall efficacy profiles that may translate to improved control or an extended control window in some situations, but all tested products provide very good to excellent control of powdery mildew on wheat.
... A number of fungicides can affect cell membrane permeability of phytopathogens (Duan et al., 2013;Zhu et al., 2019;Hou et al., 2019;Duan et al., 2019;Duan et al., 2018). Unsurprisingly, CPT was reported to affect bacterial growth and replication by causing membrane damage in A. avenae subsp. ...
Rice blast caused by Magnaporthe oryzae B. Couch is one of the most devastating diseases on rice. Camptothecin (CPT), which was primarily isolated from Camptotheca acuminata, is well-known for its anti-tumor activities, and is also developed as a potential biological pesticide. We previously investigated the anti-microbial activities of CPT against 11 fungi, 3 oomycetes, and 4 bacteria, and found that CPT was strongly effective against M. oryzae, indicating its potential as a lead for developing fungicide against rice blast. However, the anti-fungal effects of CPT on M. oryzae need further elucidation. In this study, the anti-fungal activities of CPT against M. oryzae were further investigated, which revealed that CPT was effective against M. oryzae both in vitro and in vivo. The transcriptome of M. oryzae was analyzed after CPT treatment, which showed that CPT had a strong inhibitory effect on 'translation' and 'carbohydrate metabolism/energy metabolism' of M. oryzae. Some physiology characteristics of M. oryzae were also assayed, which confirmed that CPT inhibited RNA synthesis, protein synthesis, and carbohydrate metabolism/energy metabolism of M. oryzae, and caused membrane damage. The molecular simulation result showed that CPT binds to the interface of DNA-topoisomerase I complex of M. oryzae. In conclusion, CPT is a promising lead for developing fungicide against rice blast. CPT may bind to DNA-topoisomerase I complex of M. oryzae, thus affecting 'translation' and 'carbohydrate metabolism/energy metabolism', leading to cell death.
... On the detached barley leaves, coumoxystrobin exhibited excellent protective and curative effects, and the protective effect was better than the curative effect. Similar results have also been observed with other fungicides (Duan et al., 2019c;Li et al., 2018). These findings provide references for the timing of fungicides application to control rice blast. ...
Rice blast, caused by Magnaporthe oryzae, is a destructive fungal disease in rice, causing serious losses in yield and quality. Coumoxystrobin is a novel methoxyacrylate strobilurin fungicide. In the current study, we determined the sensitivity of 100 M. oryzae strains to coumoxystrobin based on the mycelial growth inhibition method. The EC50 values ranged from 0.0089 to 0.0290 μg mL-1, with a mean EC50 value of 0.0163 ± 0.0036 μg mL-1, indicating that coumoxystrobin exhibits an excellent inhibitory activity in the mycelial growth of M. oryzae. In addition, the EC50 values had no significant difference among four populations from the different geographical regions. After treating with coumoxystrobin, cell membrane permeability increased, respiration decreased, and the hyphal tips were contorted, with offshoot of top increasing. Protective and curative activity tests showed that coumoxystrobin exhibited better protective and curative activities against M. oryzae in detached barley leaves in comparison to the currently used fungicides tricyclazole and azoxystrobin. Also, it was found that the protective activity was better than its curative activity. Furthermore, compared with the currently used fungicides, coumoxystrobin not only exhibited excellent control efficacy on rice blast, but also markedly reduced the dosages of chemical fungicides in the field trials. Overall, these findings provide important references for revealing the pharmacological effect of coumoxystrobin against M. oryzae and managing rice blast caused by M. oryzae.
Succinate dehydrogenase inhibitor (SDHI) fungicides are the most commonly and effectively used class of fungicides for controlling gray mold. Among them, only boscalid has been registered in China for controlling grape gray mold, while isofetamid and pydiflumetofen are two new SDHI fungicides that have demonstrated high efficacy against various fungal diseases. However, the sensitivity to these three fungicides against B. cinerea isolates from vineyards in China are currently unknown. In this study, the sensitivity of 55 B. cinerea isolates from vineyards to boscalid, isofetamid and pydiflumetofen were determined, with EC 50 values ranging from 1.10 to 393, 0.0300 to 42.0 and 0.0990 to 25.5 μg mL ⁻¹ , respectively. The resistance frequencies for boscalid, isofetamid and pydiflumetofen were 60.0%, 7.2% and 12.8%, respectively. Three mutations (H272R, H272Y, P225F) were detected in the SdhB subunit, with H272R being the most prevalent (75.7%), followed by H272Y (16.2%) and P225F (8.1%). All three mutations are associated with resistance to boscalid, of which H272R mutants exhibited high resistance. Only P225F and H272Y mutants exhibited resistance to isofetamid and pydiflumetofen, respectively. A weakly positive cross-resistance relationship was observed between boscalid and pydiflumetofen (r = 0.38, P < 0.05). Additionally, the H272R mutants showed no significant fitness costs, whereas the remaining mutants exhibited reduced mycelial growth (P225F) and sporulation (H272Y and P225F). These results suggest that isofetamid and pydiflumetofen are effective fungicides against B. cinerea in vineyards, but appropriate rotation strategies must be implemented to reduce the selection of existing SDHI-resistant isolates.
Sudden death syndrome (SDS), caused by Fusarium virguliforme, is an important yield-limiting disease of soybean (Glycine max). From 1996 to 2022, cumulative yield losses attributed to SDS in North America totaled over 25 million metric tons, valued at over $7.8 billion USD. Seed treatments are widely used to manage SDS by reducing early season soybean root infection by F. virguliforme. Fluopyram (SDHI - FRAC 7), a fungicide seed treatment for SDS management, has been registered for use on soybean in the U.S. since 2014. A baseline sensitivity study conducted in 2014 evaluated 130 F. virguliforme isolates collected from five U.S. states to fluopyram in a mycelial growth inhibition assay and reported a mean EC 50 of 3.35 mg/L. This baseline study provided the foundation for the objectives of this research: to detect any statistically significant change in fluopyram sensitivity over time and geographical regions within the U.S. and to investigate sensitivity to the fungicide pydiflumetofen. We repeated fluopyram sensitivity testing on a panel of 80 historical F. virguliforme isolates collected from 2006-2013 (76 of which were used in the baseline study) and conducted testing on 123 contemporary isolates collected from 2016-2022 from eleven U.S. states. This study estimated a mean absolute EC 50 of 3.95 mg/L in isolates collected from 2006-2013 and a mean absolute EC 50 of 4.19 mg/L in those collected in 2016-2022. There was no significant change in fluopyram sensitivity (P = 0.1) identified between the historical and contemporary isolates. A subset of 23 isolates, tested against pydiflumetofen under the same conditions, estimated an mean absolute EC 50 of 0.11 mg/L. Moderate correlation was detected between fluopyram and pydiflumetofen sensitivity estimates (R = 0.53, P < 0.001). These findings enable future fluopyram and pydiflumetofen resistance monitoring and inform current soybean SDS management strategies in a regional and national context.
Fungicides have played a key role in crop disease control worldwide. However, resistance development in pathogen populations became a serious concern in as early as 1970s and has increased further in recent years. More stringent pesticide regulation based on precautionary principle has made the introduction of new chemistry challenging. Despite such difficulties, many fungicides have been developed successfully and some of them have novel mode of actions such as the inhibition of DHODH and GWT1. In this paper, the author reviews large numbers of literature on newly developed fungicides in relation to resistance. The approach to mitigate resistance which includes the application of non-fungitoxic disease resistance inducers, crop resistance breeding, and biocontrol is also discussed briefly.
Gray mold, caused by Botrytis cinerea, poses a significant threat to the strawberry industry worldwide. B. cinerea is a high-risk pathogen in the sense of fungicide resistance. The sensitivities of B. cinerea isolates collected from Zhejiang Province, China, to the succinate dehydrogenase inhibitors (SDHIs) boscalid and Y12196 were determined based on discriminatory dose or 50% effective concentration (EC 50 ). Of the 42 isolates collected in 2018, 15 were resistant to boscalid (35.7%), and 3 were resistant to Y12196 (7.1%). Among the 84 isolates collected in 2019, the EC 50 values for boscalid ranged from 0.097 to 54.162 mg/L, while the EC 50 values for Y12196 ranged from 0.284 to 20.147 mg/L. Sequence analysis showed that the B. cinerea isolates carrying P225F (proline → phenylalanine) and N230I (asparagine → isoleucine) mutations in SDH subunit B exhibited cross-resistance between boscalid and Y12196. However, boscalid-resistant isolates with a point mutation at position 272 of SDH B (H272R, histidine → arginine) were more sensitive to Y12196. Consistent with this, Y12196, but not boscalid, could successfully inhibit the growth of B. cinerea carrying the H272R mutation (BcSDH B−H272R ) on detached strawberries and leaves. Molecular docking simulations further revealed that the hydrogen bonds and π-π interactions were formed between Y12196 and BcSDH B−H272R , but not between boscalid and BcSDH B−H272R , explaining why the boscalid-resistant B. cinerea H272R mutant isolates were sensitive to Y12196. Together, our results suggested that Y12196 could effectively control boscalid resistance associated with the H272R mutation.
Sclerotinia sclerotiorum, a necrotrophic fungal plant pathogen, causes serious economic losses on a wide variety of hosts. In this study, the current sensitivity of S. sclerotiorum toward the new type QoI fungicide pyribencarb and morphological and biochemical effects of pyribencarb on this pathogen were investigated. The mean EC50 value for 100 isolates collected in 2014 was 0.0379 ± 0.0163 μg/mL with individual EC50 values ranging from 0.0068 to 0.0883 μg/mL, and the EC50 values were normally distributed. Fluorescence microscopic analysis revealed pyribencarb in PDA at 0.10 µg/mL had no obvious effects on the number of the cell nucleus or the development of septum of S. sclerotiorum. The result of light microscopy indicated that pyribencarb could make hyphae of S. sclerotiorum contorted and the offshoots increased. Pyribencarb at 0.02 and 0.10 µg/mL significantly increased exopolysaccharide content and reduced cell membrane permeability and oxalic acid content (P < 0.05). There was no significant difference in the number (P = 0.75) or dry weight (P = 0.67) of sclerotia between the control and the treatment with pyribencarb at concentrations from 0.0125 to 0.2 µg/mL. The observed mycelial growth inhibitions for the mixtures of pyribencarb at 0.02 µg/mL and Congo red at 1 to 4 mg/mL or sodium dodecyl sulfate at 0.0025 to 0.02% were higher than the expected inhibitions. The protective and curative efficacies of pyribencarb were significantly higher (P < 0.004) than that of the reference fungicide carbendazim, and protective efficacy was superior to curative efficacy. Our findings suggest that pyribencarb has a remarkable impact on the morphological and biochemical characteristics of S. sclerotiorum thus advancing our understanding of the mode of action of pyribencarb on phytopathogenic fungi.
Fusarium crown rot (FCR) caused by Fusarium pseudograminearum is one of the most important diseases impacting wheat production in the Huanghuai region, the most important wheat-growing region of China. The current study found that the SDHI fungicide pydiflumetofen, which was recently developed by Syngenta Crop Protection, provided effective control of 67 wild-type F. pseudograminearum isolates in artificial culture (PDA), with an average EC50 value of 0.060 ± 0.0098 μg/mL (SE). Further investigation revealed that the risk of fungicide resistance in pydiflumetofen was not especially high. Four F. pseudograminearum mutants generated by repeated exposure to pydiflumetofen under the laboratory conditions indicated that pydiflumetofen resistance was associated with fitness penalties. Mutants exhibited significantly (p < 0.05) reduced sporulation in mung bean broth, as well as significantly (p < 0.05) reduced pathogenicity in wheat seedlings. Sequence analysis indicated that the observed pydiflumetofen resistance of the mutants was likely associated with amino acid changes in the different subunits of the succinate dehydrogenase target protein, including R18L and V160M substitutions in the FpSdhA sequence, as well as D69V, D147G, S199S, and C257R in FpSdhB, and W78R in FpSdhC. This study found no evidence of cross-resistance between pydiflumetofen and the alternative fungicides tebuconazole, fludioxonil, carbendazim, or fluazinam, which all have distinct modes of action and could therefore be used in combination or rotation with pydiflumetofen to reduce the risk of resistance emerging in the field. Taken together, these results indicate that pydiflumetofen has potential as a novel fungicide for the control of FCR caused by F. pseudograminearum, and could therefore be of great significance to ensuring high and stable wheat yields in China.
Succinate dehydrogenase inhibitors (SDHIs) are keystone synthetic fungicides used to manage Botrytis cinerea in several hosts. In this study, we investigated the cross-resistance between five new SDHIs, including pyraziflumid, isofetamid, benzovindiflupyr, fluxapyroxad, and pydiflumetofen with commonly used SDHIs, boscalid and fluopyram. Different SdhB mutations were detected in a B. cinerea collection from Michigan grapes, and their frequency and EC50 value were determined. Among 216 B. cinerea isolates, five different mutations were detected, including H272R/Y, P225F/H, and N230I at frequencies 82.6, 4.3, 11.5, 0.4, and 5.3 %, respectively. Five isolates of each genotype were used to screen the cross-resistance of the SDHIs. We have classified the resistant profile of our mutants into five patterns. We report that all tested mutants were sensitive to benzovindiflupyr, indicating that it can be used as an effective fungicide against all B. cinerea mutants. In addition, fluopyram, pydiflumetofen, and isofetamid can provide effective control according to which type of mutation is present in the field. We also developed and compared two molecular diagnostic tools, rhAMP and TaqMan assays, for rapid detection of SDHI resistance-associated mutants in B. cinerea. We report that the TaqMan assay was more successful than the rhAMP assay in detecting the B. cinerea mutant DNA at ≤ 10 pg, and in a single assay was capable of monitoring two amino acids positions. Our results provide essential information about new SDHIs and provide molecular tools for monitoring SDHI resistance mutations, which will assist in gray mold disease control.
To control apple scab caused by Venturia inaequalis, fungicides inhibiting the growth of fungal pathogens during the initial stages of infection are important management tools. From 2019 to 2021, bioassays to evaluate the curative effects of fungicides typically used to control apple scab revealed that iminoctadine triacetate, inpyrfluxam, pydiflumetofen, ipflufenoquin, fluopyram, fluxapyroxad, and cyprodinil exerted curative effects against V. inaequalis. Overall, iminoctadine triacetate, inpyrfluxam, and pydiflumetofen were particularly effective. Control of primary infection is important for the management of scab. Therefore, these curative fungicides should be applied during the period from half-inch green to fruit set.
Twenty‐one novel 5‐trifluoromethyl‐1H‐pyrazole‐4‐carboxamide derivatives were designed, synthesized, and evaluated for their in vitro anticancer and antifungal activities; some exhibited good anticancer activity against cancer cells and antifungal activity against fungi. The IC50 values of T1, T4, T6, and T7 against A549 cells were 24.9, 21.9, 14.0, and 10.2 μM, respectively, and those of T15 against Thanatephorus cucumeris and Botryosphaeria dothidea were 22.0 and 27.7 μg/mL, respectively. A morphological study using FM and SEM demonstrated that T15 destroys the integrity and permeability of the T. cucumeris cell membrane. The results of molecular docking between T15 and SDH (PDB code: 2FBW) indicated that amide and trifluoromethyl might be the key functional groups for target compounds with high fungicidal activities.
Peanut stem rot caused by Sclerotium rolfsii is a serious soil-borne disease and poses a threat to the peanut production. The antibiotic fungicide tetramycin has a broad antifungal spectrum against multiple pathogens and possess low environmental risks. In current study, a total of 250 isolates collected from Huanghuai peanut-growing region of China (Henan, Shandong and Hebei Province) were used to establish the baseline sensitivity of S. rolfsii to tetramycin. The baseline sensitivity curve was unimodal and distributed from 0.01 to 0.36 mg/L, with a mean EC50 (50% effective concentration) value of 0.11 ± 0.06 mg/L. Tetramycin also had strong inhibitory activity on the formation and germination of sclerotia. There was no significant correlation of S. rolfsii sensitivity to tetramycin and other commonly used SDHI (succinate dehydrogenase inhibitor), QoI (quinone outside respiration inhibitor) and DMI (demethylation inhibitor) fungicides. Moreover, tetramycin significantly increased the cell membrane permeability and reduced the oxalate acid content. Greenhouse experiments showed that tetramycin has both protective and curative efficacy against S. rolfsii, while protective efficacy was higher than curative efficacy. Anyhow, the bioactivity of tetramycin is similar (curative efficacy) or higher (protective efficacy) than the control fungicide validamycin. In terms of application method, root drench may be more suitable for tetramycin than spraying, because root drench of tetramycin obtained a higher efficacy. These results indicated that tetramycin may be a potential alternative fungicide for the efficient control of peanut stem rot.
Tomato gray mold (TGM), a worldwide destructive disease, is caused by Botrytis cinerea, and fungicide treatments are effective for its control. Pydiflumetofen is a new fungicide of succinate dehydrogenase inhibitors. In this research, 153 B. cinerea isolates were collected from eight different cities in Hebei Province from 2015 to 2017. The sensitivity of these 153 isolates to pydiflumetofen was determined by spore germination assays and mycelial growth rate assays. The results indicated that for these tested isolates, the EC50 values of pydiflumetofen ranged from 0.0036 to 0.0822 mg/L (on conidia germination), and 0.0287–1.2659 mg/L (on mycelial growth rate), with average EC50 values of 0.0327 ± 0.0213 mg/L and 0.3717 ± 0.2744 mg/L, respectively. The frequency distribution of the sensitivity of these 153 isolates to pydiflumetofen was a continuous single-peak curve, and could be used as the baseline for testing the sensitivity of B. cinerea to pydiflumetofen. No cross-resistance existed between pydiflumetofen and the other five botryticides such as diethofencarb, pyrimethanil, iprodione, pyrisoxazole, and fludioxonil, apart from fluopyram. The control efficacy of pydiflumetofen against TGM was determined using detached leaves and in field trials. Pydiflumetofen exhibited good control efficacy against TGM and the preventive effect was better than the curative effect. In the field trials, the control efficacy of pydiflumetofen against TGM was higher than 80%, both on leaves and fruits, at doses of 120, 160 and 200 g a.i./ha. It was significantly better than pyrimethanil. This study showed that pydiflumetofen had high activity and could effectively control the prevalence of TGM, but it should be administered carefully.
Mediator is a nucleus-localized, multi-subunit protein complex highly conserved across eukaryotes. It interacts with RNA polymerase II transcription machinery as well as various transcription factors to regulate gene expression. However, systematic characterization of the Mediator complex has not been performed in filamentous fungi. In our study, the goal was to investigate key biological functions of Mediator subunits in a mycotoxigenic plant pathogen Fusarium verticillioides. While there is some level of divergence in the constituent subunits, the overall structure was conserved between Saccharomyces cerevisiae and F. verticillioides. We generated 11 Mediator subunit deletion mutants and characterized vegetative growth, conidia formation, environmental stress response, carbon and fatty acid utilization, virulence and fumonisin B1 (FB1) biosynthesis. Each Mediator subunit deletion mutant showed deficiencies in at least three of the phenotypes tested, suggesting that each subunit has different principal functions in F. verticillioioides development, metabolism, and virulence. The deletion of FvMed1 led to increased FB1 production, and we confirmed that FvMed1 is transported from the nucleus to the cytoplasm under fumonisin-producing conditions. Taken together, our study characterized various important functional roles for Mediator subunits in F. verticillioides and suggests that select subunits can perform unique cytoplasmic functions independent of the core Mediator in fungal nucleus.
Pydiflumetofen, a fungicide of the class of succinate dehydrogenase inhibitors, can disrupt energy metabolism by inhibiting the synthesis of succinate dehydrogenase, thus effectively inhibiting pathogenic fungal growth and related yield losses.We studied the adsorption and desorption behaviors and interaction mechanisms of pydiflumetofen in eight different arable soils by the infrared spectroscopy and batch equilibrium method. Pydiflumetofen adsorption and desorption property of soils conformed with the Freundlich isotherm model and the values for the adsorption capacity KF-ads were in the range of 14.592–102.610. The adsorption constants (KF-ads) exhibited a significantly positive and linear correlation (p < 0.1) with soil organic matter and organic carbon content. Both high and low temperatures weakened the pydiflumetofen sorption capacity of the soil. In addition, the initial pH of the solution, its ionic strength, and the addition of exogenous biochar, humic acid, and different types of surfactants at different concentrations also affected the sorption property of the soil. Pydiflumetofen is weakly mobile and leachable in most soils, and, poses some threat to surface soil and water organisms, but does not contaminate groundwater.
Oilseed rape (OSR; Brassica napus) is a globally important crop which is increasingly under pressure from pests, pathogens and weeds. We investigated the potential of achieving multifunctional crop protection benefits by intercropping oilseed rape with legumes. A field experiment was conducted in which winter oilseed rape was intercropped with the annual frost sensitive legumes berseem clover (Trifolium alexandrinum) or spring faba bean (Vicia faba), or with the winter grain legumes winter faba bean or winter peas (Pisum sativum). We tracked damage to winter oilseed rape by autumn and spring pests (slugs and insects), pathogens, weed biomass, as well as oilseed rape and intercrop yield in each treatment. Intercropping treatments resulted in pest damage that was equivalent or lower than in oilseed rape alone. Follow up field and lab assessments for the frost sensitive legume intercrops provided evidence for a reduction in autumn pest damage to OSR. Each legume intercrop had its own benefits and drawbacks in relation to pest, pathogen and weed suppression, suggesting that the plant species selected for intercropping with oilseed rape should be based on the pests, pathogens and weeds of greatest concern locally to achieve relevant multifunctional benefits. Our study provides a framework for further experiments in which the multifunctional effects of intercropping on pests, pathogens and weeds can be quantified.
Succinate dehydrogenase inhibitors (SDHIs) have become one of the fastest growing classes of new fungicides since entering the market, and have attracted increasing attention as a result of their unique structure, high activity and broad fungicidal spectrum. The mechanism of SDHIs is to inhibit the activity of succinate dehydrogenase, thereby affecting mitochondrial respiration and ultimately killing pathogenic fungi. At present, they have become popular varieties researched and developed by major pesticide companies in the world. In the review, we focused on the mechanism, the history, the representative varieties, structure–activity relationship and resistance of SDHIs. Finally, the potential directions for the development of SDHIs were discussed. It is hoped that this review can strengthen the individuals’ understanding of SDHIs and provide some inspiration for the development of new fungicides.
Pydiflumetofen is a broad-spectrum fungicide, the residues of which contaminate crops or are released into the environment, posing a threat to organisms and human health. In this study, pydiflumetofen residues in paddy field ecosystems were determined using quick, easy, cheap, rugged, safe (QuECHERS) sample preparation coupled to high-performance liquid chromatography–mass spectrometry (HPLC–MS) analysis. The dissipation of pydiflumetofen residues was investigated in four Chinese paddy fields over a two-year period (2019 and 2020). The results show that pydiflumetofen dissipation in paddy water occurs faster than those in rice straw and paddy soil, with half-lives of 0.72–2.47, 1.09–9.34, and 6.08–14.38 d, respectively. Linear analysis revealed that the dissipation half-life of pydiflumetofen in different matrices is positively correlated with soil organic matter content and pH and negatively correlated with rainfall and temperature. The final residues of pydiflumetofen in brown rice were determined to assess its dietary-intake risk, and through the household method of soaking for 2-4h, 28.65-40.24% pydiflumetofen residue can be removed from the rice. The acute and chronic dietary exposure risks of pydiflumetofen in rice were found to be 4.57%–7.14% (acute hazard index) and 0.58%–2.09% (hazard quotient), respectively, indicating that pydiflumetofen poses little or no health risk to Chinese consumers. These results will help guide the practical application of pydiflumetofen and help minimize the environmental risks associated with its global use.
The necrotrophic pathogen Sclerotinia sclerotiorum is one of the most damaging and economically important plant pathogens. Pydiflumetofen, which was recently developed by Syngenta Crop Protection, has already been registered in China for the management of Sclerotinia stem rot (SSR) which was caused by S. sclerotiorum in oilseed rape. In an attempt to preempt and forestall the development of resistance to this useful fungicide, the current study was initiated to investigate potential mechanism of resistance in laboratory mutants. Five pydiflumetofen-resistant S. sclerotiorum mutants were successfully generated by repeated exposure to the fungicide under laboratory conditions. Although the mutants had greatly reduced sensitivity to pydiflumetofen, they were also found to have significantly (p < 0.05) reduced fitness exhibiting reduced mycelial growth and sclerotia formation on PDA medium. However, three of the four mutants had significantly (p < 0.05) increased pathogenicity on detached soybean leaves compared to their respective parental isolates, indicating a moderate to high level of fungicides resistance risk according to the criteria of Fungicide Resistance Action Committee (FRAC). Sequence analysis of four succinate dehydrogenase (Sdh) target genes identified several nucleotide changes in the sequences of the pydiflumetofen-resistant mutants, most of which were synonymous and caused no changes to the predicted amino acid sequences. However, all of the pydiflumetofen-resistant mutants had two amino acid point mutations (A11V and V162A) in their predicted SsSdhB sequence. No similar changes were found in the SsSdhA, SsSdhC, and SsSdhD genes of any of the mutants tested. In addition, there was a positive cross-resistance between pydiflumetofen and boscalid, and no cross-resistance between pydiflumetofen and other commonly used fungicides, including tebuconazole, fludioxonil, cyprodinil, dimethachlone, prochloraz, pyraclostrobin, fluazinam, procymidone, and carbendazim. These results indicate that pydiflumetofen has great potential as an alternative fungicide for the control of S. sclerotiorum, especially where resistance to other fungicides has already emerged. Mixing or alternate application with fludioxonil, prochloraz, and fluazinam could be used to limit the risk of resistance to pydiflumetofen.
Early blight, caused by Alternaria solani, is observed annually in all midwestern potato production areas. The use of foliar fungicides remains a primary management strategy. However, A. solani has developed reduced-sensitivity or resistance to many single-site fungicides such as quinone outside inhibitor (QoI, FRAC group 11), succinate dehydrogenase inhibitor (SDHI, FRAC group 7), demethylation inhibitors (DMI, FRAC group 3), and anilinopyrimidine (AP, FRAC group 9) fungicides. Boscalid, fluopyram, solatenol, and adepidyn are EPA-registered SDHI fungicides used commercially on a variety of crops, including potato. Five SDH mutations have been characterized previously in A. solani that affect the efficacy of boscalid while only one of these mutations has been demonstrated to negatively affect fluopyram efficacy. Conidial germination assays were used to determine if a shift in sensitivity has occurred in these SDHI fungicides. Alternaria solani isolates collected prior to the commercial application of SDHI fungicides (baseline) and were compared to recently collected isolates (non-baseline). Greenhouse evaluations were conducted also to evaluate the efficacy of boscalid, fluopyram, solatenol, and adepidyn on A. solani isolates possessing individual SDH mutations. Additionally, field trials were conducted to determine the effects application of these SDHI fungicides on the frequency of SDH mutations. Fluopyram, solatenol, and adepidyn had high intrinsic activity against A. solani when compared to boscalid, based on in vitro assays. The application of adepidyn and solatenol resulted in greater early blight control than the application of boscalid and fluopyram in greenhouse experiments. Molecular characterization of A. solani isolates collected from the field trials determined that the frequency of the H134R-mutation can increase in response to more recently developed SDHI fungicides. In contrast, the H278R/Y- and H133R-mutations decreased to the point of being nearly absent in these field experiments.
Sclerotinia sclerotiorum is a devastating plant pathogen with a broad host range and worldwide distribution. The application of chemical fungicides is a primary strategy for controlling this pathogen. However, under the high selective pressure of chemical fungicides, fungicide resistance has emerged and gradually increased, resulting in the failure to control S. sclerotiorum in the field. Quinofumelin is a novel quinoline fungicide, but its antifungal activities against plant pathogens have been rarely reported. Here, we determined the antifungal activity of quinofumelin against S. sclerotiorum in vitro and in planta. The EC50 values ranged from 0.0004 to 0.0059 μg mL-1 with a mean EC50 of 0.0017 ± 0.0009 μg mL-1 and were normally distributed (P=0.402). In addition, no cross-resistance was observed between quinofumelin and other fungicides, dimethachlone, boscalid, or carbendazim, which are commonly used to manage S. sclerotiorum. Quinofumelin did not affect glycerol and oxalic acid production of either carbendazim-sensitive or -resistant isolates. Moreover, quinofumelin exhibited excellent protective, curative, and translaminar activity against S. sclerotiorum on oilseed rape leaves. Protective activity was higher than curative activity. Interestingly, quinofumelin inhibited the formation of the infection cushion in S. sclerotiorum, which may contribute to the control efficacy of quinofumelin against S. sclerotiorum in the field. Our findings indicate that quinofumelin has excellent control efficacy against S. sclerotiorum in vitro and in planta as compared with the currently extensively used fungicides and could be used to manage carbendazim- and dimethachlone-resistance in S. sclerotiorum in the field.
Pydiflumetofen is a new fungicide that belongs to the succinate dehydrogenase inhibitor (SDHI) class. This novel fungicide can help protect soybean plants against infection by foliar pathogens, including Cercospora sojina (causal agent of frogeye leaf spot) and Corynespora cassiicola (causal agent of target spot). A total of 88 single-spore isolates of C. sojina collected from soybean fields between 2007 and 2019 from Alabama, Georgia, Illinois, Indiana, Iowa, Kentucky, Louisiana, Michigan, Minnesota, Mississippi, Nebraska, South Dakota, and Tennessee and a total of 76 C. cassiicola single-spore isolates collected from soybean fields in Kentucky between 2017 and 2019 were evaluated for their sensitivity to pydiflumetofen. For this purpose, in vitro bioassays were conducted using different doses of pydiflumetofen (0, 0.001, 0.01, 0.1, 1, and 10 μg/ml). The effective concentrations of pydiflumetofen in which C. sojina and C. cassiicola isolates were inhibited by 50% (EC50) were calculated. The C. sojina EC50 values ranged from 0.335 to 4.335 μg/ml, the mean value was 1.616 μg/ml and median was 1.309 μg/ml. The range, mean and median of C. cassiicola EC50 were 0.037–0.248 μg/ml and 0.086 and 0.069 μg/ml, respectively. Establishing a baseline sensitivity is essential to identify any shifts in sensitivity of fungal populations over time. The findings in this current paper could be used to help monitor the sensitivity of C. sojina and C. cassiicola to pydiflumetofen over time.
Succinate dehydrogenase inhibitors (SDHI) comprise a class of broad-spectrum fungicides utilized for management of diseases caused by phytopathogenic fungi. In many cases, reduced sensitivity to SDHI fungicides has been correlated with point mutations in the SdhB and SdhC target genes that encode components of the succinate dehydrogenase complex. The genetic basis of SDHI fungicide resistance mechanisms, however, has been functionally characterized in very few fungi. Sclerotinia sclerotiorum is a fast-growing and SDHI fungicide-sensitive phytopathogenic fungus that can be conveniently transformed. Given the high amino acid sequence similarity and putative structural similarity of SDHI protein target sites between S. sclerotiorum and other common phytopathogenic ascomycete fungi, we developed an in vitro heterologous expression system that used S. sclerotiorum as a reporter strain. With this system, we were able to demonstrate the function of mutant SdhB or SdhC alleles from several ascomycete fungi in conferring resistance to multiple SDHI fungicides. In total, we successfully validated the function of Sdh alleles that had been previously identified in field isolates of Botrytis cinerea, Blumeriella jaapii, and Clarireedia jacksonii (formerly S. homoeocarpa) in conferring resistance to boscalid, fluopyram or fluxapyroxad, and used site-directed mutagenesis to construct and phenotype a mutant allele that is not yet known to exist in Monilinia fructicola populations. We also examined the functions of these alleles in conferring cross-resistance to more recently introduced SDHIs including inpyrfluxam, pydiflumetofen, and pyraziflumid. The approach developed in this study can potentially be widely applied to interrogate SDHI fungicide resistance mechanisms in other phytopathogenic ascomycetes.
Gummy Stem Blight (GSB), caused by Didymella bryoniae, is a devastating disease on watermelon. Pydiflumetofen belongs to SDHI fungicide, which is effective in controlling many plant diseases. The EC50 values of 69 D. bryoniae isolates to pydiflumetofen ranged from 0.0018 to 0.0071 μg/ml and the MIC value of all strains to pydiflumetofen was < 0.05 μg/ml. Eight pydiflumetofen-resistant mutants were obtained and the level of resistant was stable. The mycelial growth, dry weight of mycelia, hyphal morphology and pathogenicity of most resistant mutants did not change significantly compared with their parental strains, which indicated that the resistance risk of D. bryoniae to pydiflumetofen would be medium to high. Sequencing alignment showed that five resistant mutants presented a mutation at codon 277 (H277Y) in SdhB gene. The point mutants FgSdhBH248Y/R exhibited decreased sensitivity to pydiflumetofen in Fusarium graminearum, which indicated that the point mutants of SdhB could reduce sensitivity to pydiflumetofen. These results further increase our understanding about the mode of action and resistance mechanism of pydiflumetofen.
Succinate dehydrogenase inhibitor (SDHI) fungicides are currently the most frequently used fungicides for controlling gray mold. However, isolates of Botrytis cinerea resistant to SDHI fungicides have emerged in the field. Pydiflumetofen is a new SDHI fungicide that can control a variety of fungal diseases, but its efficacy against gray mold and whether the activity of pydiflumetofen is affected by the current SDHI-resistant isolates is currently unknown. The sensitivity of 291 single-spore B. cinerea isolates collected from 2017 to 2019 to pydiflumetofen was determined by spore germination inhibition assays. The mean EC50 value (fungicide concentration resulting in a 50% inhibition compared with that of the control) of pydiflumetofen was 0.06 ± 0.01, 0.07 ± 0.02, and 0.05 ± 0.02 mg/liter in 2017, 2018, and 2019, respectively. There was no significant difference in the sensitivity of B. cinerea to pydiflumetofen among the 3 years. Furthermore, pydiflumetofen at 300 mg/liter effectively controlled gray mold on cucumber leaves (80.9%), and its efficacy was superior to that of boscalid at 400 mg/liter (42.7%). The isolates carrying P225F, N230I, H272Y, and H272R mutations in the SdhB subunit were associated with the less sensitivity of B. cinerea to SDHI fungicides. After establishing the baseline sensitivity of B. cinerea to pydiflumetofen (EC50 of 0.03 ± 0.003 mg/liter), we found that the P225F and H272Y mutant isolates showed low to moderate levels of resistance to pydiflumetofen, and the H272R and N230I mutant isolates showed low levels of resistance. The reduced sensitivity to pydiflumetofen resulted from the positive correlation of pydiflumetofen with the other four SDHI fungicides (i.e., boscalid, fluopyram, isopyrazam, and benzovindiflupyr). These results suggest that pydiflumetofen provides effective control for the management of gray mold but must be used with caution.
Herein we present a facile approach to a diverse collection of 1,4-disubstituted-3-di- or mono-fluoromethylpyrazoles utilizing our previously developed cyanopyrazoles as key building blocks. This method features several merits, such as easily accessible starting materials, broad substrate scope, mild reaction conditions, and simple operation. This protocol further deserves to be highlighted by the successful translation into the synthesis of commercialized fungicide Fluxapyroxad and its analogues.
The point mutation at codon 200 (TTC -> TAC, F200Y) confers moderate resistance to carbendazim in Sclerotinia sclerotiorum. This mutant genotype (F200Y) has been detected mainly by determining the minimum inhibitory concentration (MIC), which requires 3 to 5 days. Here, we developed a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of the F200Y mutant genotype of carbendazim-resistant isolates of S. sclerotiorum. Specific LAMP primers were designed and concentrations of LAMP components were optimized. The optimal reaction conditions were 62 to 63 degrees C for 45 min. The new LAMP assay requires no special equipment and is highly sensitive and specific (the i.e., it generated positive results with F200Y mutant genotype but generated negative results with other carbendazim-resistant mutants and with a variety of carbendazim-resistant mutants of Botrytis cinerea and Fusarium graminearum). Inclusion of the loop backward (LB) primer reduced the reaction time to 15 min. Results were identical with LAMP and MIC determinations. The advantages of the LB-accelerated LAMP assay for detection of the F200Y mutant genotype were demonstrated by assaying sclerotia produced on rape stems that were artificially inoculated in the field. The results indicated that the new LAMP assay represents an improved way to detect the F200Y mutant genotype of carbendazim-resistant isolates of S. sclerotiorum.
Resistance of benzimidazole fungicides is related to the point mutation of the β-tubulin gene in Sclerotinia sclerotiorum. The point mutation at codon 198 (GAG → GCG, E198A) occurs in more than 90% of field resistant populations in China. Traditional detection methods of benzimidazole-resistant mutants of S. sclerotiorum are time-consuming, tedious and inefficient. To establish a suitable and rapid detection of benzimidazole-resistant mutants of S. sclerotiorum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). Eight sets of LAMP primers were designed and four sets were optimized to specially distinguish benzimidazole-resistant mutants of S. sclerotiorum. With the optimal LAMP primers, the concentration of LAMP components was optimized and the reaction conditions were set as 60-64 °C for 60 min. This method had a good specificity, sensitivity, stability and repeatability. In the 1491 sclerotia, 614 (41.18%) were positive by LAMP, and 629 (42.19%) positive by MIC. Therefore, the LAMP assay is more feasible to detect benzimidazole-resistant mutants of S. sclerotiorum than traditional detection methods.
Baseline sensitivity of Sclerotinia sclerotiorum to fluazinam was determined using 150 isolates collected during 2011 and 2013 from the rapeseed fields without a previous history of fluazinam usage in Jiangsu province of China. The median effective concentration (EC50) values for fluazinam inhibiting mycelial growth on agar plates ranged from 0.0019 to 0.0115 μg/mL (mean of 0.0069 μg/ml). Neither cross-resistance between fluazinam and carbendazim or dimethachlon was detected. None of the tested isolates displayed reduced sensitivity to fluazinam. On detached rapeseed leaves and pot experiments, fluazinam at 150 μg/ml provided over 80 % control efficacy and exhibited excellent protective activity. In field trials, fluazinam at 187.5 g a.i. ha−1 provided over 70 % control efficacy, higher than other traditional fungicides, suggesting excellent activity against Sclerotinia stem rot (SSR). These results suggest that fluazinam has strong antifungal activity and could be a good alternative fungicide to carbendazim or dimethachlon in controlling Sclerotinia stem rot.
Gray mold is an important disease in grapevines in Chile, and its control depends primarily on the use of fungicides with a single-site mode of action. Botrytis cinerea has a high risk of developing resistance against such fungicides. Therefore, novel chemical options are needed to achieve satisfactory control of gray mold. Isofetamid is a new succinate dehydrogenase inhibitor (SDHI) fungicide with a single-site of action that inhibits cellular respiration and appears to be a new option in the chemical treatment against gray mold. The aim of this study was to determine the effectiveness of isofetamid in controlling grapevine gray mold in Chile. Field trials undertaken in three different commercial vineyards showed that isofetamid controls gray mold to a similar extent as fenhexamid. During 2012 and 2013, 10 B. cinerea isolates were obtained from commercial vineyards in central Chile to determine their sensitivity to isofetamid. The median inhibitory concentration of isofetamid varied between 0.3-10.0 µg mL-1 and 0.6 to >10 µg mL-1 for mycelium and conidia, respectively. The efficacy of isofetamid against B. cinerea in apple bioassays varied between 61-100% and 37.5-100% for mycelium and conidia, respectively. The results of this study demonstrate that isofetamid is a highly effective fungicide against B. cinerea. © 2014, Pontificia Universidad Catolica de Chile, Facultad de Agronomia e Ingenieria Forestal. All Rights reserved.
Stem rots caused by Rhizoctonia solani and Sclerotinia sclerotiorum have been known as devastating diseases in balloon flower plants. Antifungal activities of four fungicides, azoxystrobin, polyoxin B, trifloxystrobin and validamycin A were evaluated in vitro, showing effective suppression with mycelial growth of the fungal isolates on PDA media. Efficacies of the four fungicides were also demonstrated in stem tissues of balloon flower plants against R. solani and S. sclerotiorum. A commercially available Bacillus subtilis strain Y1336 was tested in terms of antagonistic biological control of stem rot disease of balloon flower plants. The bacterial strain revealed its antifungal activities against R. solani and S. sclerotiorum demonstrated by dual culture tests using paper discs and two plant pathogenic fungi on PDA media, as well as by plant inoculation assay, indicating that this antagonistic bacterial strain can be incorporated into disease management program for balloon flower stem rot diseases together with the four chemical fungicides.
Sclerotinia sclerotiorum is a necrotrophic, phytopathogenic, filamentous ascomycete with a broad host range and a worldwide distribution. Application of fungicides is the principal tool for controlling Sclerotinia diseases on most crops. Unfortunately, the extensive use of a single fungicide selects for resistant populations and leads to control failures. The phenylpyrrole fungicide fludioxonil has been reported to have high activity against S. sclerotiorum and to control Sclerotinia stem rot in rapeseed. In this study, biochemical characteristics of laboratory-induced mutants of S. sclerotiorum were determined. The results indicated that the fludioxonil-resistant mutants were sensitive to osmotic stress (high sugar). Compared to the wild-type strains, the fludioxonil-resistant mutants had a significant increase in cell membrane permeability, glycerol and oxalate content, and phenylalanine ammonia-lyase and peroxidase activity, but did not differ in exopolysaccharide content. Sequencing indicated that three wild-type strains were identical, and the mutants SZ45R and HA61R had a single point mutation while NT18R had both a single point mutation and a frameshift in the amino acid sequence coded by the two-component histidine kinase gene (Shk1, SS1G_12694). Therefore, we concluded that the biological differences between the resistant mutants and the wild-type strains may be related to mutation in Shk1. The information will increase our understanding of the resistance mechanism of S. sclerotiorum to fludioxonil and could provide new reference data for the management of Sclerotinia stem rot caused by S. sclerotiorum.
Protoplast transformation is an important technique for establishing a mutation library and determining gene function for Sclerotinia sclerotiorum and other plant pathogenic fungi. In this study, we determined the effect of various conditions on preparation of protoplasts for transformation. These conditions included the age of the culture providing the hyphae to be digested; enzyme composition, buffer solution and concentration; and digestion time and temperature. The optimum conditions for preparing protoplasts were as follows: 10 ml of enzyme solution (1.5% lysing enzyme in 0.8 m mannitol and citric acid-sodium citrate buffer) reacting with 0.1 g of hyphae (cultured for 36 h) at 30°C for 2.5 h. The transformation efficiency was 60–85 transformants per microgram of DNA. In addition, an expression vector for gene complementation was constructed, and an additional dominant selectable marker (neomycin) was demonstrated. To verify the reliability of the expression vector, we constructed and transformed the complementation vector of Shk1 for gene complementation based on the Shk1 deletion mutant △Shk1. The results showed that the expression level and biological phenotypes of Shk1 were restored in the complementary strain △Shk1+Shk1. The techniques and procedures described will improve our ability to study gene function in S. sclerotiorum and are likely applicable to other plant pathogens.
The necrotrophic fungal phytopathogen Sclerotinia sclerotiorum (Lib.) de Bary has a broad host range and frequently causes destructive diseases. The extensive use of common fungicides to control these diseases has selected for resistance in populations of S. sclerotiorum. In this study, 105 isolates of S. sclerotiorum from different geographical regions in Jiangsu Province of China were characterized for baseline sensitivity to azoxystrobin, and the average EC50 value was 0.2932 μg/mL for mycelial growth. Of the mixtures of the fungicides thiram and azoxystrobin that were tested using an in vitro mycelial growth assay, the 1:4 ratio provided the greatest inhibition of S. sclerotiorum. When tested against nine isolates, the 1:4 mixture resulted in a mean synergy ratio of 2.31, indicating synergistic inhibition. Mycelial respiration was inhibited for about 2 h by azoxystrobin alone but for 48 h by the mixture of thiram and azoxystrobin. Salicylhydroxamic acid (SHAM, a known inhibitor of alternative respiration) also increased the inhibition of mycelial growth and respiration caused by azoxystrobin. These results suggest the need for further study of effects of combinations of azoxystrobin with thiram or SHAM in planta to evaluate their potential for management of diseases caused by S. sclerotiorum.
Fungal histidine kinases (HKs) are involved in osmotic and oxidative stress responses, hyphal development, fungicide sensitivity and virulence. Members of HK class III are known to signal through the high-osmolarity glycerol mitogen-activated protein kinase (HOG MAPK). In this study, we characterized the Shk1 gene (SS1G_12694.3), which encodes a putative class III HK, from the plant pathogen Sclerotinia sclerotiorum. Disruption of Shk1 resulted in resistance to phenylpyrrole and dicarboximide fungicides and increased sensitivity to hyperosmotic stress and H2 O2 -induced oxidative stress. The Shk1 mutant showed a significant reduction in vegetative hyphal growth and was unable to produce sclerotia. Quantitative real-time polymerase chain reaction (qRT-PCR and glycerol determination assays showed that the expression of SsHOG1 (the last kinase of the Hog pathway) and glycerol accumulation were regulated by the Shk1 gene, but PAK (p21-activated kinase) was not. In addition, the Shk1 mutant showed no change in virulence. All the defects were restored by genetic complementation of the Shk1 deletion mutant with the wild-type Shk1 gene. These findings indicate that Shk1 is involved in vegetative differentiation, sclerotial formation, glycerol accumulation and adaption to hyperosmotic and oxidative stresses, and to fungicides, in S. sclerotiorum. Taken together, our results demonstrate, for the first time, the role of two-component HKs in Sclerotinia.
Fusarium head blight (FHB) or scab mainly caused by Fusarium asiaticum is a devastating fungal disease in wheat. In China, carbendazim (MBC) and its co-formulated mixture with other fungicides are the main fungicides for controlling the disease. But resistant populations have developed severely in the field. The fungicides for controlling the disease are limited. So screening novel fungicides for controlling FHB is urgent. The fungicide pydiflumetofen (development code number: SYN545974),3-(Difluoromethyl)N-methyl-N-[1-methyl-2-(2,4,6-trichlorophenyl)ethyl]-1H-pyrazole-4-carboxami.d.e, is a novel succinate dehydrogenase inhibitor (SDHI) that was developed by Syngenta. In this study, the baseline sensitivities for different isolates of F. asiaticum from various regions of China to pydiflumetofen were established. The 50% effective concentration (EC50) value of pydiflumetofen on suppressing mycelial growth against 116 isolates of F. asiaticum was 0.019–0.2084 μg/ml and their average value was 0.0745 ± 0.0367 μg/ml. The EC50 value of pydiflumetofen on suppressing conidium germination was 0.0583–0.4237 μg/ml and their average value was 0.1813 ± 0.0861 μg/ml. The results indicated that the fungicide had strong inhibition effect on mycelial growth and conidium germination against F. asiaticum in vitro. The EC50 values distribution of 116 isolates measured by mycelial growth assay or by conidium germination assay was a unimodal curve and could be used for detecting any sensitivity changes of F. asiaticum populations to pydiflumetofen in the future. The results of cross-resistance analysis indicated that there was no positive or negative cross-resistance between pydiflumetofen and carbendazim/phenamacril. In field trials, though FHB was very severe, pydiflumetofen at 125, 150 and 200 g ai ha⁻¹ all provided more than 80% control efficacy which was significantly higher than that of phenamacril at 375 g ai ha⁻¹ or MBC at 600 g ai ha⁻¹ in 2015 and 2016. In conclusion, pydiflumetofen had very high activity against F. asiaticum in vitro and excellent efficacy for controlling FHB could also manage carbendazim-resistant F. asiaticum populations in the field.
Background:
Net blotch caused by Pyrenophora teres is an important disease of barley worldwide. In addition to strobilurins (QoIs) and azoles (DMIs), succinate dehydrogenase inhibitors (SDHIs) are very effective fungicides for net blotch control. Recently, SDHI resistant isolates have been found in the field. Intensive sensitivity monitoring programmes across Europe were carried out to investigate the situation of SDHI resistance in P. teres.
Results:
The first isolates with a lower sensitivity to SDHIs registered in barley were found in Germany in 2012 and carried the B-H277Y substitution in succinate dehydrogenase enzyme. In 2013 and 2014 a significant increase of isolates with lower SDHI sensitivity was detected mainly in France and Germany and the range of target site mutations increased. Most of the resistant isolates carried the C-G79R substitution which exhibits a strong impact on all SDHIs in microtiter tests. All SDHIs tested were shown to be cross-resistant. Other substitutions are gaining in importance, e.g. C-N75S in France and D-D145G in Germany. So far, no double mutants in SDH genes have been detected. Glasshouse tests showed that SDHI resistant isolates were still controlled by the SDHI fluxapyroxad when applied preventative. To date, most isolates with C-G79R substitution did not simultaneously carry the F129L change in cytochrome b, which causes resistance towards QoI fungicides at low to moderate levels.
Conclusion:
Several target site mutations in the genes of subunits SDH-B, SDH-C and SDH-D with different impact on SDHI fungicides were detected. The pattern of mutations varied from year to year and between different regions. Strict resistance management strategies are recommended to maintain SDHIs as effective tools for net blotch control, especially in areas with low frequencies of resistant isolates.
Oxalate secretion by fungi provides many advantages for their growth and colonization of substrates. The role of oxalic acid in pathogenesis is through acidification of host tissues and sequestration of calcium from host cell walls. The formation of calcium oxalate crystals weakens the cell walls, thereby allowing polygalacturonase to effect degradation more rapidly in a synergistic response. There is good correlation between pathogenesis, virulence, and oxalic acid secretion. Solubility of soil nutrients is achieved by soil-living species, when cations freed by oxalate diffusing in clay layers increases the effective solubility of Al and Fe. Oxalate retained in hyphal mats of mycorrhizal species increases phosphate and sulphate availability. The formation of calcium oxalate crystals provides a reservoir of calcium in the ecosystem. The ability of oxalate to bind divalent cations permits detoxification of copper, particularly evident in wood preserved with copper salts. Oxalate plays a unique role in lignocellulose degradation by wood-rotting basidiomycetes, acting as a low molecular mass agent initiating decay. In addition, in white-rot fungi oxalate acts as a potential electron donor for lignin-peroxidase catalysed reduction and chelates manganese, allowing the dissolution of Mn3+ from the manganese-enzyme complex and thus stimulating extracellular manganese peroxidase activity. The biosynthesis and degradation of oxalate are discussed.
Sclerotinia stem rot caused by Sclerotinia sclerotiorum, is the main fungal disease of oilseed rape (Brassica napus) in Jiangsu Province of China, causing oil quality declines and yield losses from 10 to 80 %. Chemical control, such as carbendazim (MBC) and dimethachlon, remains the major method to reduce the incidence of S. sclerotiorum. Isolates collected from Sclerotinia-infected stems of oilseed crops in Jiangsu province between 2010 and 2012, were tested for their sensitivity to carbendazim and dimethachlon in order to determine the level of resistance to these fungicides in the population of S. sclerotiorum in that province. Sensitivity tests showed that 375 isolates were resistant to carbendazim among the 3,701 isolates with a resistance frequency of 10.1 %; only one isolate in the Lian Yungang region of Jiangsu Province in 2010 showed normal growth at 5 μg/ml dimethachlon. The proportion of carbendazim-resistant isolates ranged from 0 to 44 % in different regions of Jiangsu. The field MBC-resistant isolates showed comparable mycelial growth, sclerotial production, pathogenicity and osmotic sensitivity to the MBC-sensitive isolates, which suggested that the field MBC-resistant isolates might have sufficient parasitic fitness to compete with the field sensitive isolates in the field; whereas the dimethachlon-resistant isolates were less fit than their parental isolates in terms above. Moreover, S. sclerotiorum exhibited positive cross-resistance between dimethachlon and other dicarboximide fungicides such as iprodione and procymidone.
SYP-7017, a broad-spectrum fungicide, belongs to the chemical group of strobilurins. Baseline sensitivity of Sclerotinia sclerotiorum (Lib) de Bary to SYP-7017 was determined using 120 strains collected during 2010 and 2011 from rapeseed fields without a previous history of strobilurin usage in Jiangsu Province, China. The median effective concentration (EC50) values for SYP-7017 that inhibited mycelial growth ranged from 0.006 to 0.047 µg mL−1 (mean of 0.016 µg mL−1). No cross-resistance between SYP-7017 and carbendazim or iprodione was detected. On detached rapeseed leaves, SYP-7017 at 100 µg mL−1 provided over 90% control efficacy. Moreover, SYP-7017 exhibited good characteristics of absorption and translocation, and also exhibited excellent protective activity in pot experiments. In field trials, control efficacies of SYP-7017 at 75 g a.i. ha−1 and 187.5 g a.i. ha−1 were 80.2% and 91.58%, respectively, higher than other traditional fungicides. These results suggest that SYP-7017 has strong antifungal activity and a potential application in controlling S. sclerotiorum.
Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is the main fungal disease of oilseed rape (Brassica napus) in China. Numerous fungicide applications are required for control. Dimethachlon, one of the dicarboximide fungicides, has been the major fungicide for disease control after benzimidazole resistance became widespread. Fungal populations were collected throughout Jiangsu Province between 2006 and 2007 in order to determine their sensitivity to dimethachlon. A total of 1,066 single-sclerotium isolates of S. sclerotiorum were collected, and most of the isolates were considered sensitive to dimethachlon. Five isolates collected in Yancheng and Changzhou showed normal growth at 5 mu g/ml dimethachlon with the resistance factor approximate to 10 (resistance factor was estimated as ratios between the EC(50) values of resistant isolates and the average EC(50) values of sensitive ones) compared to the sensitive isolates (EC(50) is the concentration of fungicide causing 50% reduction in growth). Through in vitro selection for resistance to the fungicide, 25 dimethachlon-resistant mutants were derived from 10 wild-type isolates of S. sclerotiorum. The resistance factors for the isolates ranged from 198 to 484, and the isolates were considered highly resistant to dimethachlon. Therefore, at least two different mechanisms of resistance seem to be involved: one that may provide a moderate resistance (insensitivity) and a second that may give a high resistance level under laboratory conditions. There was positive cross-resistance between dimethachlon and other dicarboximide fungicides, such as iprodione and procymidone, in these S. sclerotiorum isolates. The field dimethachlon-insensitive and the laboratory-induced dimethachlon-resistant isolates appeared to have mycelial growth, sclerotial production, and pathogenicity comparable to their wild-type parental isolates. Also, results of osmotic tests showed that there were no significant difference in mycelial radial growth between the field dimethachlon-sensitive and field dimethachlon-insensitive isolates on potato dextrose agar plates amended with 2, 4, 6, or 8% (wt/vol) NaCl, but the laboratory-induced dimethachlon-resistant isolates grew significantly more slowly than their wild-type sensitive parents under all concentrations of NaCl. Because these studies yielded a high frequency of laboratory resistance in S. sclerotiorum. together with the occurrence of field insensitivity, appropriate precautions against resistance development in natural populations should be taken.
Fungicide and biological control treatments were evaluated for efficacy in suppressing white mold of bean in six field trials. In all trials, treatment with the fungicide benomyl significantly suppressed disease. In almost all trials, at least one biological control treatment significantly suppressed disease but no biological control treatments provided significant suppression in more than two trials. Stability analyses of the treatment responses in the different environments (e.g., trials) indicated that most treatments responded in a linear manner as environmental conditions became more conducive to disease. All treatments became less effective as conditions became more conducive for disease but the fungicide treatment provided a relatively stable suppression of disease in all tested environments. The biological control treatments provided a relatively unstable suppression of disease and, in general, were effective only when environmental conditions were less favorable for disease development. The results establish that all of the evaluated disease management practices were responsive to environmental factors present during epidemics of white mold and indicate why biological controls often provide a more variable suppression of disease than fungicides.
Between 2006 and 2008, 120 isolates of Sclerotinia sclerotiorum from the Jiangsu Province of China were characterized for the baseline sensitivity to boscalid, a new active ingredient that interferes with succinate ubiquinone reductase in the electron transport chain of fungi. The isolates collected between 2006 and 2008 all showed similar sensitivity to boscalid. Baseline sensitivities were distributed as unimodal curves with mean EC50 values of 0.17±0.09μg ml−1 for inhibition of mycelial growth. Inhibition of mycelial growth of isolates whether they had resistance to carbendazim or, dimethachlon or dual resistance to both carbendazim and dimethachlon was similar, suggesting that there was no cross-resistance between boscalid and MBC/dimethachlon already in commercial use. In field trials, the treatments boscalid (50% WG), at 125, 187.5 and 250g ai ha−1 provided higher control efficacies of 65.2, 69.0 and 75.1%, respectively, than the treatments carbendazim (50% WP), at 750g ai ha−1 and dimethachlon (46% WP), at 690g ai ha−1 with control efficacies of 18.9 and 44.6%, respectively.
Recombinant plasmids pIJ3040 and pIJ3041 containing overlapping fragments of the genome of wild type Xanthomonas campestris pv. campestris restored the production of extracellularpolysaccharide to non-slimy mutants. Mutagenesis of the cloned DNA with the transposon Tn5 was used to localize the complementing region of the DNA. Marker-exchange of Tn5 insertions from cloned DNA into the X.c. campestris genome provided evidence for linked genes involved in EPS production. the EPS-deficient mutants retained pathogenicity in a seedlin bioassay.
An index of plants reported to be susceptible to Sclerotinia sclerotiomm was compiled from the scientific literature. The index contains 42 subspecies or varieties, 408 species, 278 genera and 75 families. Except for one species in the Pteridophyta, all hosts of S. sclerotiomm occur in the classes Gymnospermae and Angiospermae of the division Spermatophyta. Most hosts are herbaceous plants from the subclass Dicotyledonae of the Angiospermae but several hosts also occur in the subclass Monocotyledonae. The organization in this index of our knowledge of the host specificity of S. sclerotiomm may facilitate the control of diseases caused by this pathogen and the use of the fungus as an agent of biological control.
Oxalate secretion by fungi provides many advantages for their growth and colonization of substrates. The role of oxalic acid in pathogenesis is through acidification of host tissues and sequestration of calcium from host cell walls. The formation of calcium oxalate crystals weakens the cell walls, thereby allowing polygalacturonase to effect degradation more rapidly in a synergistic response. There is good correlation between pathogenesis, virulence, and oxalic acid secretion. Solubility of soil nutrients is achieved by soil-living species, when cations freed by oxalate diffusing in clay layers increases the effective solubility of Al and Fe. Oxalate retained in hyphal mats of mycorrhizal species increases phosphate and sulphate availability. The formation of calcium oxalate crystals provides a reservoir of calcium in the ecosystem. The ability of oxalate to bind divalent cations permits detoxification of copper, particularly evident in wood preserved with copper salts. Oxalate plays a unique role in lignocellulose degradation by wood-rotting basidiomycetes, acting as a low molecular mass agent initiating decay. In addition, in white-rot fungi oxalate acts as a potential electron donor for lignin-peroxidase catalysed reduction and chelates manganese, allowing the dissolution of Mn3+ from the manganese–enzyme complex and thus stimulating extracellular manganese peroxidase activity. The biosynthesis and degradation of oxalate are discussed.Key words: oxalic acid, calcium oxalate, pathogenicity, fungi.
Sclerotinia sclerotiorum (Lib.) de Bary and Sclerotinia minor Jagger are two of the species in the genus Sclerotinia found in Canada. While S. sclerotiorum is distributed country-wide and causes disease on numerous economically important crops including oilseeds, pulses, forage legumes, vegetables, and ornamentals, S. minor has only been reported on lettuce in Quebec and Ontario. During the past five decades, Canadian researchers have made a substantial contribution to the understanding of the biology and control of S. sclerotiorum and, to a lesser extent, of S. minor. This review summarizes the progress of Canadian research on Sclerotinia diseases in the past 50 years, with specific emphasis in the areas such as strain differentiation, epidemiology, new types of sclerotia, survival and germination of sclerotia, and biocontrol.
Le Sclerotinia sclerotiorum (Lib.) de Bary et le Sclerotinia minor Jagger sont deux espèces du genre Sclerotinia trouvées au Canada. Alors que le S. sclerotiorum a une distribution pancanadienne et cause des maladies sur de nombreuses cultures d'importance économique, y compris des plantes oléagineuses, des légumineuses alimentaires, des légumineuses fourragères, des légumes et des plantes ornementales, le S. minor n'a été rapporté que sur la laitue au Québec et en Ontario. Au cours des cinq dernières décennies, les chercheurs canadiens ont contribué de façon substantielle à la compréhension de la biologie et de la lutte contre le S. sclerotiorum et, dans une moindre mesure, contre le S. minor. La présente synthèse résume les progrès accomplis par la recherche canadienne sur les sclérotinioses, au cours des derniers 50 ans, en mettant spécifiquement l'accent sur des domaines tel que la différenciation des souches, l'épidémiologie, les nouveaux types de sclérotes, la survie et la germination des sclérotes et la lutte biologique.
This work determined the sensitivity of field populations of Sclerotinia sclerotiorum (Lib.) de Bary before exposure to the fungicide fludioxonil (= baseline sensitivity) and assessed the risk of fludioxonil resistance. The mean EC(50) (Effective Concentration) and Minimum inhibitory concentration (MIC) values for fludioxonil based on inhibition of mycelial growth of 120 wild-type isolates were 0.015 +/- 0.005 mu g/ml and <0.05 mu g/ml, respectively. Positive cross-resistance was not detected between fludioxonil and benzimidazole fungicides but was detected between fludioxonil and dicarboximide fungicides which are considered as high resistance risk fungicides by FRAC, even though these fungicides have different molecular structures. By growing wild-type isolates on potato dextrose agar (PDA) containing sublethal concentrations of the fungicide, we obtained four fludioxonil-resistant mutants with resistance factors (EC(50) resistant/EC(50) sensitive phenotypes) >2000. The laboratory fludioxonil mutants were less fitter than their parental isolates in terms of mycelial radial growth, pathogenicity and sclerotial production. Moreover, on PDA amended with NaCl, the laboratory fludioxonil mutants grew more slowly than their fludioxonil-sensitive parents, especially at lower concentrations of NaCl. According to the fitness of mutants and the cross-resistance between fludioxonil and dicarboximide fungicides, phenylpyrroles can be considered to pose a moderate resistance risk. In a field trial, fludioxonil provided greater control (over 90% disease control) of S. sclerotiorum than iprodione. (C) 2011 Published by Elsevier Ltd.
Carbendazim (MBC) was widely used to control Sclerotinia stem rot routinely during the 1980s in China, but development of
MBC resistance in the causal agent Sclerotinia sclerotiorum led to control failures of this disease. In this study it was found that the MBC resistance in S. sclerotiorum populations was widespread throughout Jiangsu Province with a resistance frequency of 29.54% in the 1786 collected isolates
during the growing seasons of 2006 to 2008. The resistance frequencies differed among sampled cities, ranging from 3.1% to
54.9%. The field MBC-resistant isolates showed comparable mycelial growth, sclerotia production and pathogenicity to the wild-type
sensitive isolates, which suggested that the field MBC-resistant isolates might have sufficient parasitic fitness to compete
with the field MBC-sensitive isolates in the field. In the in vitro sensitivity test, boscalid showed greater activity against S. sclerotiorum than dicarboximide fungicides (dimethachlon, iprodione and procymidone). The treatment 50% boscalid (WG) 125g a.i. ha−1 was comparable in efficacy to the treatment 50% iprodione (WP) 600g a.i. ha−1, and better than other treatments of 6% dimethachlon (WP) 690g a.i. ha−1 and 50% procymidone (WP) 337.5g a.i. ha−1, whereas MBC failed to control Sclerotinia stem rot (control efficacy only 16.0%). The most active agent for controlling
Sclerotinia stem rot was boscalid in our study.
The role of oxalic acid in pathogenicity of the bean white mould fungus, Sclerotinia sclerotiorum, was investigated. Prototrophic mutants of the fungus deficient in oxalic acid production were obtained by UV irradiation of ascospores. Deficiency in oxalic acid production was screened by colour change on potato dextrose agar containing the indicator dye bromophenol blue. An enzymatic method, gas chromotography and high performance liquid chromatography indicated that the selected mutants did not produce oxalic acid in carbohydrate-rich media or bean blossoms, a natural substrate. In growth chamber experiments using whole plants, stems and leaves and in laboratory tests using pods, these acid minus mutants were nonpathogenic while the oxalic acid-producing wild type and revertant were pathogenic. Production of oxalic acid was induced when mutants were grown on nutrient media containing sodium succinate. Mutants grown on this medium and inoculated on bean leaves penetrated and incited small lesions. No association between pathogenicity and pectolytic enzymes was found. These studies present confirming evidence that oxalic acid is a pathogenicity determinant.
Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic fungal pathogen causing disease in a wide range of plants. This review summarizes current knowledge of mechanisms employed by the fungus to parasitize its host with emphasis on biology, physiology and molecular aspects of pathogenicity. In addition, current tools for research and strategies to combat S. sclerotiorum are discussed.
Taxonomy:Sclerotinia sclerotiorum (Lib.) de Bary: kingdom Fungi, phylum Ascomycota, class Discomycetes, order Helotiales, family Sclerotiniaceae, genus Sclerotinia.
Identification: Hyphae are hyaline, septate, branched and multinucleate. Mycelium may appear white to tan in culture and in planta. No asexual conidia are produced. Long-term survival is mediated through the sclerotium; a pigmented, multi-hyphal structure that can remain viable over long periods of time under unfavourable conditions for growth. Sclerotia can germinate to produce mycelia or apothecia depending on environmental conditions. Apothecia produce ascospores, which are the primary means of infection in most host plants.
Host range:S. sclerotiorum is capable of colonizing over 400 plant species found worldwide. The majority of these species are dicotyledonous, although a number of agriculturally significant monocotyledonous plants are also hosts.
Disease symptoms: Leaves usually have water-soaked lesions that expand rapidly and move down the petiole into the stem. Infected stems of some species will first develop dark lesions whereas the initial indication in other hosts is the appearance of water-soaked stem lesions. Lesions usually develop into necrotic tissues that subsequently develop patches of fluffy white mycelium, often with sclerotia, which is the most obvious sign of plants infected with S. sclerotiorum.
Useful websites:http://www.whitemoldresearch.com; http://www.broad.mit.edu/annotation/fungi/sclerotinia_sclerotiorum
Rhizobial plant symbionts and bacterial plant pathogens produce exopolysaccharides that often play essential roles in the plant interaction. Many of these exopolysaccharides are acidic heteropolysaccharides that have repeating subunit structures with carbohydrate and noncarbohydrate substituents, while others are homopolysaccharides such as alginate, levan, cellulose, and glucan. While the homopolysaccharides are synthesized by mechanisms that vary with the particular polysaccharide, the heteropolysaccharides as a rule are synthesized by subunit assembly from nucleotide diphosphate-sugar precursors on a membrane-bound lipid carrier followed by polymerization and secretion. Many mutants in exopolysaccharide synthesis have been isolated, and in several cases this has led to the identification of genes that function in particular steps of biosynthesis, as well as in regulation of exopolysaccharide biosynthesis. The genetic regulation of exopolysaccharide synthesis in many plant pathogens is complex, perhaps reflecting the various niches, free living and in planta, in which exopolysaccharides function. In some cases, exopolysaccharide synthesis is regulated coordinately with other virulence factors, and in other cases separately. Regulatory genes that have homology to the two-component sensor and transcriptional effector systems are a common motif. In Rhizobium species, exopolysaccharide synthesis is regulated by transcriptional as well as posttranslational mechanisms. Exopolysaccharides function differently in the root-nodule symbiosis versus plant pathogenesis. Specific Rhizobium exopolysaccharide structures promote nodule development and invasion in legumes that form indeterminate nodules. In plant pathogenesis, less specific mechanisms of pathogenesis occur: exopolysaccharides cause wilting by blocking xylem vessels, are partly responsible for water-soaked lesions, and may also aid in invasion, growth, and survival in plant tissues.
Three exopolysaccharide (EPS)- and virulence-deficient mutants of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, were isolated by Tn5 mutagenesis. These insertions are not located within the gum gene cluster. A 40-kb cosmid clone that restored EPS production and virulence to all three mutants was isolated, and the three transposon insertions were localized to contiguous 4.3- and 3.5-kb EcoRI fragments that are included in this clone. Sequence data indicate that two of the transposon insertions are in genes that encode a putative sugar nucleotide epimerase and a putative glycosyl transferase, respectively; the third insertion is located between the glycosyl transferase gene and a novel open reading frame (ORF). A 5.5-kb genomic region in which these three ORFs are located has a G+C content of 5-1.7%, quite different from the G+C content of approximately 65.0% that is typical of X. oryzae pv. oryzae. Homologues of this locus have not yet been reported in any other xanthomonad.
ADEPIDYN (TM) fungicide: Cross resistance patterns in Alternaria solani
- G Olaya
- R Linley
- K Edlebeck
- T Harp
Olaya, G., Linley, R., Edlebeck, K., and Harp, T. 2016. ADEPIDYN (TM)
fungicide: Cross resistance patterns in Alternaria solani. Phytopathology (suppl.)
106:S4.12.
Emergence of succinate dehydrogenase inhibitor resistance of
- A Rehfus
- S Miessner
- J Achenbach
- D Strobel
- R Bryson
- G Stammler
Rehfus, A., Miessner, S., Achenbach, J., Strobel, D., Bryson, R., and Stammler, G.
409
2016. Emergence of succinate dehydrogenase inhibitor resistance of
Adepidyn (TM): A new fungicide active ingredient for control of foliar diseases
- K Buxton
- T Harp
- A Tally
- H Mclean
Buxton, K., Harp, T., Tally, A., and Mclean, H. 2016. Adepidyn (TM): A new
fungicide active ingredient for control of foliar diseases. Phytopathology
(suppl.) 106:S4.61.
Adepidyn (TM): A new fungicide active ingredient for control of Fusarium head blight on wheat
- T Harp
- K Anaka
Harp, T., Anaka, K., and Tally, A. 2016. Adepidyn (TM): A new fungicide active
ingredient for control of Fusarium head blight on wheat. Phytopathology
(suppl.) 106:S4.61.
Mutation in b-tubulin of Sclerotinia sclerotiorum conferring resistance to carbendazim in rapeseed field isolates
- H X Li
- Y J Lu
- M G Zhou
Li, H. X., Lu, Y. J., and Zhou, M. G. 2003. Mutation in b-tubulin of Sclerotinia
sclerotiorum conferring resistance to carbendazim in rapeseed field isolates.
Chin. J. Oil Crop Sci. 2:56-60.