Characterization of the fludioxonil and phenamacril dual resistant mutants of Fusarium graminearum.

Fusarium graminearum is an important fungal pathogen causing Fusarium head blight (FHB) in wheat and other cereal crops worldwide. Due to lack of resistant wheat cultivars, FHB control mainly relies on application of chemical fungicides. Both fludioxonil (a phenylpyrrole compound) and phenamacril (a cyanoacrylate fungicide) have been registered for controlling FHB in China, however, fludioxonil-resistant isolates of F. graminearum have been detected in field. To evaluate the potential risk of dual resistance of F. graminearum to both compounds, fludioxonil and phenamacril dual resistant (DR) mutants of F. graminearum were obtained via fungicide domestication in laboratory. Result showed that resistance of the DR mutants to both fludioxonil and phenamacril were genetically stable after sub-cultured for ten generations or stored at 4 °C for 30 days on fungicide-free PDA. Cross-resistance assay showed that the DR mutants remain sensitive to other groups of fungicides, including carbendazim, tebuconazole, pydiflumetofen, and fluazinam. In addition, the DR mutants exhibited defects in mycelia growth, conidiation, mycotoxin deoxynivalenol (DON) production, and virulence Moreover, the DR mutants displayed increased sensitivity to osmotic stress. Sequencing results showed that amino acid point mutations S217L/T in the myosin I protein is responsible for phenamacril resistance in the DR mutants. Our results indicate that mutations leading to fludioxonil and phenamacril dual resistance could result in fitness cost for F. graminearum. Our results also suggest that the potential risk of F. graminearum developing resistance to both fludioxonil and phenamacril in field could be rather low, which provides scientific guidance in controlling FHB with fludioxonil and phenamacril.

Comparative transcriptome analysis provides insights into the resistance regulation mechanism and inhibitory effect of fungicide phenamacril in Fusarium asiaticum.

Fusarium asiaticum is a destructive phytopathogenic fungus that causes Fusarium head blight of wheat (FHB), leading to serious yield and economic losses to cereal crops worldwide. Our previous studies indicated that target-site mutations (K216R/E, S217P/L, or E420K/G/D) of Type I myosin FaMyo5 conferred high resistance to phenamacril. Here, we first constructed one sensitive strain H1S and three point mutation resistant strains HA, HC and H1R. Then we conducted comparative transcriptome analysis of these F. asiaticum strains after 1 and 10 µg·mL-1 phenamacril treatment. Results indicated that 2135 genes were differentially expressed (DEGs) among the sensitive and resistant strains. The DEGs encoding ammonium transporter MEP1/MEP2, nitrate reductase, copper amine oxidase 1, 4-aminobutyrate aminotransferase, amino-acid permease inda1, succinate-semialdehyde dehydrogenase, 2, 3-dihydroxybenzoic acid decarboxylase, etc., were significantly up-regulated in all the phenamacril-resistant strains. Compared to the control group, a total of 1778 and 2097 DEGs were identified in these strains after 1 and 10 µg·mL-1 phenamacril treatment, respectively. These DEGs involved in 4-aminobutyrate aminotransferase, chitin synthase 1, multiprotein-bridging factor 1, transcriptional regulatory protein pro-1, amino-acid permease inda1, ATP-dependent RNA helicase DED1, acetyl-coenzyme A synthetase, sarcoplasmic/endoplasmic reticulum calcium ATPase 2, etc., showed significantly down-regulated expression in phenamacril-sensitive strain but not in resistant strains after phenamacril treatment. In addition, cyanide hydratase, mating-type protein MAT-1, putative purine nucleoside permease, plasma membrane protein yro2, etc., showed significantly co-down-regulated expression in all the strains after phenamacril treatment. Taken together, This study provides deep insights into the resistance regulation mechanism and the inhibitory effect of fungicide phenamacril and these new annotated proteins or enzymes are worth for the discovery of new fungicide targets.

The intrinsic resistance of Fusarium solani to the Fusarium-specific fungicide phenamacril is attributed to the natural variation of both T218S and K376M in myosin5.

Fusarium solani is responsible for causing root rot in various crops, resulting in wilting and eventual demise. Phenamacril, a specific inhibitor of myosin5 protein, has gained recognition as an effective fungicide against a broad spectrum of Fusarium species. It has been officially registered for controlling Fusarium diseases through spray application, root irrigation, and seed dipping. In this study, phenamacril was observed to exhibit negligible inhibitory effects on F. solani causing crop root rot, despite the absence of prior exposure to phenamacril. Considering the high selectivity of phenamacril, this phenomenon was attributed to intrinsic resistance and further investigated for its underlying mechanism. Sequence alignment analysis of myosin5 proteins across different Fusarium species revealed significant differences at positions 218 and 376. Subsequent homology modeling and molecular docking results indicated that substitutions T218S, K376M, and T218S&K376M impaired the binding affinity between phenamacril and myosin5 in F. solani. Mutants carrying these substitutions were generated via site-directed mutagenesis. A phenamacril-sensitivity test showed that the EC50 values of mutants carrying T218S, K376M, and T218S&K376M were reduced by at least 6.13-fold, 9.66-fold, and 761.90-fold respectively compared to the wild-type strain. Fitness testing indicated that mutants carrying K376M or T218S&K376M had reduced sporulation compared to the wild-type strain. Additionally, mutants carrying T218S exhibited an enhanced virulence compared to the wild-type strain. However, there were no significant differences observed in mycelial growth rates between the mutants and the wild-type strain. Thus, the intrinsic differences observed at positions 218 and 376 in myosin5 between F. solani and other Fusarium species are specifically associated with phenamacril resistance. The identification of these resistance-associated positions in myosin5 of F. solani has significantly contributed to the understanding of phenamacril resistance mechanisms, thereby discouraging the use of phenamacril for controlling F. solani.

Phenamacril and carbendazim regulate trichothecene mycotoxin synthesis by affecting ROS levels in F. asiaticum.

Fusarium head blight caused by Fusarium asiaticum is an important cereal crop disease, and the trichothecene mycotoxins produced by F. asiaticum can contaminate wheat grain, which is very harmful to humans and animals. To effectively control FHB in large areas, the application of fungicides is the major strategy; however, the application of different types of fungicides has varying influences on the accumulation of trichothecene mycotoxins in F. asiaticum. In this study, phenamacril inhibited trichothecene mycotoxin accumulation in F. asiaticum; however, carbendazim (N-1H-benzimidazol-2-yl-carbamic acid, methyl ester) induced trichothecene mycotoxin accumulation. Additionally, phenamacril led to a lower level of reactive oxygen species (ROS) by inducing gene expression of the catalase and superoxide dismutase (SOD) pathways in F. asiaticum, whereas carbendazim stimulated ROS accumulation by inhibiting gene expression of the catalase and SOD pathways. Based on these results, we conclude that phenamacril and carbendazim regulate trichothecene mycotoxin synthesis by affecting ROS levels in F. asiaticum.

Impact of Phenamacril on the Growth and Development of Fusarium pseudograminearum and Control of Crown Rot of Wheat.

Fusarium pseudograminearum is the dominant pathogen causing Fusarium crown rot (FCR) of wheat. Phenamacril is a 2-cyanoacrylate fungicide, having a control effect on diseases caused by Fusarium spp. The objective of this study was to investigate the inhibitory effect of phenamacril on F. pseudograminearum and its control efficacy against FCR. The results showed that phenamacril had a strong inhibitory effect on the mycelial growth of F. pseudograminearum, EC50 values of phenamacril to 63 tested strains were in the range of 0.0998 to 0.5672 µg/ml, and the average EC50 value was 0.3403 ± 0.0872 µg/ml and could be used as the baseline sensitivity of F. pseudograminearum to phenamacril. Phenamacril reduced the germination rate of conidia of F. pseudograminearum, and the EC50 value was 5.0273 to 26.4814 µg/ml. In addition, we found that phenamacril had a teratogenic effect on conidia and blastotubules, which increased the ratio of conidial germination from the middle cells and showed high efficacy on the sporulation quantity of F. pseudograminearum with an EC50 value in the range of 0.0770 to 0.1064 µg/ml. There was no significant correlation between the sensitivity of F. pseudograminearum to phenamacril and its sensitivity to fludioxonil, carbendazim, tebuconazole, and kresoxim-methyl. In vitro and greenhouse assays showed that the treatment with 0.125 µl of active ingredient per gram recorded the best control effect on wheat crown rot, reaching 87.8 and 77.3%, respectively. In two experimental sites in Luoyang, phenamacril also had great control effect against FCR, reaching 83.9%. It was proven that phenamacril has a superior control effect against FCR. This study has laid a foundation for the study of the mechanism of action of phenamacril against F. pseudograminearum and provided a theoretical basis for the application of phenamacril to control FCR.

PCR-RFLP for Detection of Fusarium graminearum Genotypes with Resistance to Phenamacril.

Phenamacril is a cyanoacrylate fungicide that provides excellent control of Fusarium head blight (FHB) or wheat scab, which is caused predominantly by Fusarium graminearum and F. asiaticum. Previous studies revealed that codon mutations of the myosin-5 gene of Fusarium spp. conferred resistance to phenamacril in in vitro lab experiments. In this study, PCR restriction fragment length polymorphism (RFLP) was developed to detect three common mutations (A135T, GCC to ACC at codon 135; S217L, TCA to TTA at codon 217; and E420K, GAA to AAA at codon 420) in F. graminearum induced by fungicide domestication in vitro. PCR products of 841 bp (for mutation of A135T), 802 bp (for mutation of S217L), or 1,649 bp (for mutation of E420K) in the myosin-5 gene were amplified by appropriate primer pairs. Restriction enzyme KpnI, TasI, or DraI was used to distinguish phenamacril-sensitive and -resistant strains with mutation genotypes of A135T, S217L, and E420K, respectively. KpnI digested the 841-bp PCR products of phenamacril-resistant strains with codon mutation A135T into two fragments of 256 and 585 bp. In contrast, KpnI did not digest the PCR products of sensitive strains. TasI digested the 802-bp PCR products of phenamacril-resistant strains with codon mutation S217L into three fragments of 461, 287, and 54 bp. In contrast, TasI digestion of the 802-bp PCR products of phenamacril-sensitive strains resulted in only two fragments of 515 and 287 bp. DraI digested the 1,649-bp PCR products of phenamacril-resistant strains with codon mutation E420K into two fragments of 932 and 717 bp, while the PCR products of phenamacril-sensitive strains was not digested. The three genotypes of resistance mutations were determined by analyzing electrophoresis patterns of the digestion fragments of PCR products. The PCR-RFLP method was evaluated on 48 phenamacril-resistant strains induced by fungicide domestication in vitro and compared with the conventional method (mycelial growth on fungicide-amended agar). The accuracy of the PCR-RFLP method for detecting the three mutation genotypes of F. graminearum resistant to phenamacril was 95.12% compared with conventional method. Bioinformatics analysis revealed that the PCR-RFLP method could also be used to detect the codon mutations of A135T and E420K in F. asiaticum.

Phenamacril is a reversible and noncompetitive inhibitor of Fusarium class I myosin.

The cyanoacrylate compound phenamacril (also known as JS399-19) is a recently identified fungicide that exerts its antifungal effect on susceptible Fusarium species by inhibiting the ATPase activity of their myosin class I motor domains. Although much is known about the antifungal spectrum of phenamacril, the exact mechanism behind the phenamacril-mediated inhibition remains to be resolved. Here, we describe the characterization of the effect of phenamacril on purified myosin motor constructs from the model plant pathogen and phenamacril-susceptible species Fusarium graminearum, phenamacril-resistant Fusarium species, and the mycetozoan model organism Dictyostelium discoideum Our results show that phenamacril potently (IC50 ∼360 nm), reversibly, and noncompetitively inhibits ATP turnover, actin binding during ATP turnover, and motor activity of F. graminearum myosin-1. Phenamacril also inhibits the ATPase activity of Fusarium avenaceum myosin-1 but has little or no inhibitory effect on the motor activity of Fusarium solani myosin-1, human myosin-1c, and D. discoideum myosin isoforms 1B, 1E, and 2. Our findings indicate that phenamacril is a species-specific, noncompetitive inhibitor of class I myosin in susceptible Fusarium sp.

A New Mutation Genotype of K218T in Myosin-5 Confers Resistance to Phenamacril in Rice Bakanae Disease in the Field.

In 2017 and 2018, a total of 294 Fusarium fujikuroi isolates were collected from bakanae-diseased rice plants in Jinhua, Shaoxing, and Jiaxing in Zhejiang Province, China. Phenamacril sensitivity of these isolates was determined by the 50% effective concentration value or minimum inhibitory concentration methods. Our results indicated that the phenamacril resistance frequency of F. fujikuroi increased from 18% in 2017 to 47% in 2018, and rice plants infected with F. fujikuroi-resistant isolates could not be protected effectively with 50 mg/liter of phenamacril. Phenamacril-resistant F. fujikuroi isolates obtained from rice fields showed stable resistance, because their fitness levels (i.e., mycelial growth, sporulation, and pathogenicity) were similar to the phenamacril-sensitive isolates. In addition to the point mutation at codon 219 in the myosin-5 gene that conferred resistance to phenamacril, our results also showed another point mutation at codon 218 (AAG→ACG) in myosin-5 that also conferred resistance to phenamacril. In this study, we found rapid development and persistence of diversified genotypes of phenamacril resistance, highlighting the importance of proper use of phenamacril in rice fields. Our results may also help researchers develop new fungicides or new control strategies using combinations of different fungicides in the control of phenamacril-resistant F. fujikuroi isolates.

Determination of phenamacril residues in flour and rice based on Z-Sep+ using ultra-high-performance liquid chromatography-tandem mass spectrometry.

Phenamacril is a new broad-spectrum fungicide that is commonly used for the control of fungal diseases in wheat and rice. In this study, ultra-high-performance liquid chromatography-tandem mass spectrometry was used to establish a method for analyzing the residual phenamacril in flour and rice based on the improved QuEChERS (quick, easy, cheap, effective, rugged and safe) method using Z-Sep+ as the adsorbent in the pre-treatment process. The average recovery of phenamacril in flour and rice was 82.2-96.0%, the relative standard deviation was 2.1-5.6% and the limit of quantification was 0.5 µg/kg. The accuracy and sensitivity of this method meet the requirements for residue analysis. The method was applied to commercially available flour and rice samples, and the detected concentrations of phenamacril were 0.005-0.033 mg/kg. This method provides technical support for the safety evaluation of phenamacril.

A myosin5 dsRNA that reduces the fungicide resistance and pathogenicity of Fusarium asiaticum.

Fungal resistance to fungicides is a serious challenge in crop protection. Although strategies have been found to prevent the development of fungicide resistance, rare strategy has been found to quickly reduce such resistance once it has occurred. We demonstrate that the application of dsRNAs, which inhibit the expression of the phenamacril (fungicide JS399-19) target gene-Myosin 5 (Myo5) in Fusarium, decreased F. asiaticum resistance to phenamacril and infection. RNAi molecules derived from different regions of Myo5 gene had different effects on phenamacril-resistance. Myo5-8 (one of Myo5 segments) exhibited great and stable effect on phenamacril-resistant reduction both in vivo and in vitro. Myo5 mRNA and protein were both reduced when mycelium was treated with Myo5-8 dsRNA. After a mixture of Myo5-8 dsRNA and phenamacril treatment, plants can highly control the infection of phenamacril-resistant strain. The antifungal activity of Myo5-8 dsRNA plus phenamacril effected longer than a single Myo5-8 dsRNA. In addition, no off-target sequences were found in wheat and/or other plant and animal species for Myo5-8 dsRNA sequence. Our findings suggest a new strategy for fungicide resistant reduction and for designing new fungicides to control pathogens which easily develop fungicide resistance.

Resistance risk assessment of Fusarium oxysporum f. sp. melonis against phenamacril, a myosin inhibitor.

Fusarium wilt caused by Fusarium oxysporum f. sp. melonis (FOM) is one of the most notorious seed-borne diseases worldwide. Phenamacril is a cyanoacrylate fungicide with novel chemical structure and strong inhibitive activity against FOM. To evaluate the risk of FOM developing phenamacril resistance, five phenamacril-resistant mutants with >800µgml-1 minimum inhibitory concentration were obtained by repeated exposure to the fungicide in the laboratory. Compared with the parental isolate, four of the five phenamacril-resistant mutants showed enhanced biological fitness in sporulation and virulence, but not in sensitivity to various stresses (oxidative and osmotic pressure, cell membrane and wall inhibitor). No positive cross-resistance was observed among phenamacril and the other five fungicides, including azoxystrobin, carbendazim, boscalid, fluazinam and tebuconazole. Sequencing alignment results of the myosin 5 from the five resistant mutants and the parental strain indicated that the three resistant mutants fo-2, fo-3 and fo-4 had a single point mutation (S175L), which may confer the resistance of FOM against phenamacril. Interestingly, the resistant mutant fo-4 harbored not only one mutation (S175L) at myosin 5, but also the other mutation (A52G) at ß2-tublin. Our data supported that resistance risk of Fusarium oxysporum f. sp. melonis against phenamacril was between the moderate to high level.

Biological and molecular characterization of pydiflumetofen and phenamacril dual-resistant Fusarium graminearum strains.

BACKGROUND: Fusarium head blight (FHB) caused by Fusarium graminearum species complex (FGSG) remains a major challenge to cereal crops and resistance to key fungicides by the pathogen threatens control efficacy. Pydiflumetofen, a succinate dehydrogenase inhibitor, and phenamacril, a cyanoacrylate fungicide targeting myosin I, have been applied to combat this disease. Nonetheless, emergence of pydiflumetofen resistance in a subset of field isolates alongside laboratory-induced facile generation of phenamacril-resistant isolates signals a critical danger of resistance proliferation. RESULTS: Our study investigates the development of dual resistance to these fungicides in F. graminearum. Utilizing pydiflumetofen-resistant (PyR) and -sensitive (PyS) isolates, we obtained dual-resistant (PyRPhR) and phenamacril-resistant (PySPhR) mutants on potato sucrose agar containing phenamacril. Mutation rates for phenamacril resistance were comparable between pydiflumetofen-resistant and -sensitive isolates, implying independent pathways for resistance development. The mutants compromised in fungal growth, competitive viability and deoxynivalenol production, suggesting fitness penalties for the dual-resistant mutants. However, no cross-resistance was found with tebuconazole or fludioxonil. In addition, we characterized four critical amino acid changes (S217L, C423R, K537T, E420G) in the Myo1 that were verified to confer phenamacril resistance in F. graminearum. CONCLUSION: This research indicates the possibility of resistance development for both pydiflumetofen and phenamacril in F. graminearum and emphasizes the need for fungicide resistance management for FHB. © 2024 Society of Chemical Industry.

Unveiling the Mechanism of Phenamacril Resistance in F. graminearum: Computational and Experimental Insights into the C423A Mutation in FgMyoI.

Phenamacril (PHA) is a highly selective fungicide for controlling fusarium head blight (FHB) mainly caused by F. graminearum and F. asiaticum. However, the C423A mutation in myosin I of F. graminearum (FgMyoI) leads to natural resistance to PHA. Here, based on the computational approaches and biochemical validation, we elucidate the atomic-level mechanism behind the natural resistance of F. graminearum to the fungicide PHA due to the C423A mutation in FgMyoI. The mutation leads to a rearrangement of pocket residues, resulting in increased size and flexibility of the binding pocket, which impairs the stable binding of PHA. MST experiments confirm that the mutant protein FgMyoIC423A exhibits significantly reduced affinity for PHA compared to wild-type FgMyoI and the nonresistant C423K mutant. This decreased binding affinity likely underlies the development of PHA resistance in F. graminearum. Conversely, the nonresistant C423K mutant retains sensitivity to PHA due to the introduction of a strong hydrogen bond donor, which facilitates stable binding of PHA in the pocket. These findings shed light on the molecular basis of PHA resistance and provide new directions for the creation of new myosin inhibitors.

Comparative uptake, translocation and metabolism of phenamacril in crops under hydroponic and soil cultivation conditions.

Many studies investigate the plant uptake and metabolism of xenobiotics by hydroponic experiments, however, plants grown in different conditions (hydroponic vs. soil) may result in different behaviors. To explore the potential differences, a comparative study on the uptake, translocation and metabolism of the fungicide phenamacril in crops (wheat/rice) under hydroponic and soil cultivation conditions was conducted. During 7-14 days of exposure, the translocation factors (TFs) of phenamacril were greatly overestimated in hydroponic-wheat (3.6-5.2) than those in soil-wheat systems (1.1-2.0), with up to 3.3 times of difference between the two cultivation systems, implying it should be cautious to extrapolate the results obtained from hydroponic to field conditions. M-144 was formed in soil pore water (19.1-29.9 µg/L) in soil-wheat systems but not in the hydroponic solution in hydroponics; M-232 was only formed in wheat shoots (89.7-103.0 µg/kg) under soil cultivation conditions, however, it was detected in hydroponic solution (20.1-21.2 µg/L), wheat roots (146.8-166.0 µg/kg), and shoots (239.2-348.1 µg/kg) under hydroponic conditions. The root concentration factors (RCFs) and TFs of phenamacril in rice were up to 2.4 and 3.6 times higher than that in wheat for 28 days of the hydroponic exposure, respectively. These results highlighted that cultivation conditions and plant species could influence the fate of pesticides in crops, which should be considered to better assess the potential accumulation and transformation of pesticides in crops.

Fluorescent and Colorimetric Dual-Readout Immunochromatographic Assay for the Detection of Phenamacril Residues in Agricultural Products.

The fungicide phenamacril has been employed to manage Fusarium and mycotoxins in crops, leading to persistent residues in the environment and plants. Detecting phenamacril is pivotal for ensuring environmental and food safety. In this study, haptens and artificial antigens were synthesized to produce antiphenamacril monoclonal antibodies (mAbs). Additionally, gold nanoparticles coated with a polydopamine shell were synthesized and conjugated with mAbs, inducing fluorescence quenching in quantum dots. Moreover, a dual-readout immunochromatographic assay that combines the positive signal from fluorescence with the negative signal from colorimetry was developed to enable sensitive and precise detection of phenamacril within 10 min, achieving detection limits of 5 ng/mL. The method's reliability was affirmed by using spiked wheat flour samples, achieving a limit of quantitation of 0.05 mg/kg. This analytical platform demonstrates high sensitivity, outstanding accuracy, and robust tolerance to matrix effects, making it suitable for the rapid, onsite, quantitative screening of phenamacril residues.

Transcriptomic Profiling of Fusarium pseudograminearum in Response to Carbendazim, Pyraclostrobin, Tebuconazole, and Phenamacril.

Fusarium pseudograminearum has been identified as a significant pathogen. It causes Fusarium crown rot (FCR), which occurs in several major wheat-producing areas in China. Chemical control is the primary measure with which to control this disease. In this study, transcriptome sequencing (RNA-Seq) was used to determine the different mechanisms of action of four frequently used fungicides including carbendazim, pyraclostrobin, tebuconazole, and phenamacril on F. pseudograminearum. In brief, 381, 1896, 842, and 814 differentially expressed genes (DEGs) were identified under the carbendazim, pyraclostrobin, tebuconazole, and phenamacril treatments, respectively. After the joint analysis, 67 common DEGs were obtained, and further functional analysis showed that the ABC transported pathway was significantly enriched. Moreover, FPSE_04130 (FER6) and FPSE_11895 (MDR1), two important ABC multidrug transporter genes whose expression levels simultaneously increased, were mined under the different treatments, which unambiguously demonstrated the common effects. In addition, Mfuzz clustering analysis and WGCNA analysis revealed that the core DEGs are involved in several critical pathways in each of the four treatment groups. Taken together, these genes may play a crucial function in the mechanisms of F. pseudograminearum's response to the fungicides stress.

Insight into the uptake, accumulation, and metabolism of the fungicide phenamacril in lettuce (Lactuca sativa L.) and radish (Raphanus sativus L.).

The fungal species Fusarium can cause devastating disease in agricultural crops. Phenamacril is an extremely specific cyanoacrylate fungicide and a strobilurine analog that has excellent efficacy against Fusarium. To date, information on the mechanisms involved in the uptake, accumulation, and metabolism of phenamacril in plants is scarce. In this study, lettuce and radish were chosen as model plants for a comparative analysis of the absorption, accumulation, and metabolic characteristics of phenamacril from a polluted environment. We determined the total amount of phenamacril in the plant-water system by measuring the concentrations in the solution and plant tissues at frequent intervals over the exposure period. Phenamacril was readily taken up by the plant roots with average root concentration factor ranges of 60.8-172.7 and 16.4-26.9 mL/g for lettuce and radish, respectively. However, it showed limited root-to-shoot translocation. The lettuce roots had a 2.8-12.4-fold higher phenamacril content than the shoots; whereas the radish plants demonstrated the opposite, with the shoots having 1.5 to 10.0 times more phenamacril than the roots. By the end of the exposure period, the mass losses from the plant-water systems reached 72.0% and 66.3% for phenamacril in lettuce and radish, respectively, suggesting evidence of phenamacril biotransformation. Further analysis confirmed that phenamacril was metabolized via hydroxylation, hydrolysis of esters, demethylation, and desaturation reactions, and formed multiple transformation products. This study furthers our understanding of the fate of phenamacril when it passes from the environment to plants and provides an important reference for its scientific use and risk assessment.

Construction and characterization of ethyl cellulose-based nano-delivery system for phenamacril.

Fungicide-resistant Fusarium has become a threaten to wheat production. Novel fungicide formulations can improve the efficacy of active ingredient and minimize the emergence of resistance. Encapsulation of fungicides in biodegradable carriers, especially, in polysaccharide, is a feasible approach to develop environment-friendly and efficient formulation. This study focused on the synthesis of ethyl cellulose-based phenamacril nano-delivery system by combining emulsion-solvent evaporation and high-pressure homogenization technology to improve the control of fusarium head blight in wheat. Emulsifier 125 and Tersperse 2500 were screened from eleven commonly used surfactants. Emulsifier 125 and Tersperse 2500 in a ratio of 2:1 and phenamacril nanocapsules with the mean particle size of 152.5 ± 1.3 nm were prepared. These showed excellent storage stability and wettability on crop leaves. A bioassay comparing the nanocapsules with a commercial preparation against Fusarium graminearum showed significantly improved biological activity. This formulation could be used to effectively not only to control fusarium head blight but also delay the occurrence of resistance.

The plasma membrane H+ -ATPase FgPMA1 regulates the development, pathogenicity, and phenamacril sensitivity of Fusarium graminearum by interacting with FgMyo-5 and FgBmh2.

Fusarium graminearum, as the causal agent of Fusarium head blight (FHB), not only causes yield loss, but also contaminates the quality of wheat by producing mycotoxins, such as deoxynivalenol (DON). The plasma membrane H+ -ATPases play important roles in many growth stages in plants and yeasts, but their functions and regulation in phytopathogenic fungi remain largely unknown. Here we characterized two plasma membrane H+ -ATPases: FgPMA1 and FgPMA2 in F. graminearum. The FgPMA1 deletion mutant (∆FgPMA1), but not FgPMA2 deletion mutant (∆FgPMA2), was impaired in vegetative growth, pathogenicity, and sexual and asexual development. FgPMA1 was localized to the plasma membrane, and ∆FgPMA1 displayed reduced integrity of plasma membrane. ∆FgPMA1 not only impaired the formation of the toxisome, which is a compartment where DON is produced, but also suppressed the expression level of DON biosynthetic enzymes, decreased DON production, and decreased the amount of mycelial invasion, leading to impaired pathogenicity by exclusively developing disease on inoculation sites of wheat ears and coleoptiles. ∆FgPMA1 exhibited decreased sensitivity to some osmotic stresses, a cell wall-damaging agent (Congo red), a cell membrane-damaging agent (sodium dodecyl sulphate), and heat shock stress. FgMyo-5 is the target of phenamacril used for controlling FHB. We found FgPMA1 interacted with FgMyo-5, and ∆FgPMA1 showed an increased expression level of FgMyo-5, resulting in increased sensitivity to phenamacril, but not to other fungicides. Furthermore, co-immunoprecipitation confirmed that FgPMA1, FgMyo-5, and FgBmh2 (a 14-3-3 protein) form a complex to regulate the sensitivity to phenamacril and biological functions. Collectively, this study identified a novel regulating mechanism of FgPMA1 in pathogenicity and phenamacril sensitivity of F. graminearum.

Risk assessment and molecular mechanism of Fusarium incarnatum resistance to phenamacril.

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.