Cell-Wall-Degrading Enzymes-Related Genes Originating from Rhizoctonia solani Increase Sugar Beet Root Damage in the Presence of Leuconostoc mesenteroides.

Sugar beet crown and root rot caused by Rhizoctonia solani is a major yield constraint. Root rot is highly increased when R. solani and Leuconostoc mesenteroides co-infect roots. We hypothesized that the absence of plant cell-wall-degrading enzymes in L. mesenteroides and their supply by R. solani during close contact, causes increased damage. In planta root inoculation with or without cell-wall-degrading enzymes showed greater rot when L. mesenteroides was combined with cellulase (22 mm rot), polygalacturonase (47 mm), and pectin lyase (57 mm) versus these enzymes (0-26 mm), R. solani (20 mm), and L. mesenteroides (13 mm) individually. Carbohydrate analysis revealed increased simpler carbohydrates (namely glucose + galactose, and fructose) in the infected roots versus mock control, possibly due to the degradation of complex cell wall carbohydrates. Expression of R. solani cellulase, polygalacturonase, and pectin lyase genes during root infection corroborated well with the enzyme data. Global mRNAseq analysis identified candidate genes and highly co-expressed gene modules in all three organisms that might be critical in host plant defense and pathogenesis. Targeting R. solani cell-wall-degrading enzymes in the future could be an effective strategy to mitigate root damage during its interaction with L. mesenteroides.

Expedient Discovery for Novel Antifungal Leads Targeting Succinate Dehydrogenase: Pyrazole-4-formylhydrazide Derivatives Bearing a Diphenyl Ether Fragment.

The pyrazole-4-carboxamide scaffold containing a flexible amide chain has emerged as the molecular skeleton of highly efficient agricultural fungicides targeting succinate dehydrogenase (SDH). Based on the above vital structural features of succinate dehydrogenase inhibitors (SDHI), three types of novel pyrazole-4-formylhydrazine derivatives bearing a diphenyl ether moiety were rationally conceived under the guidance of a virtual docking comparison between bioactive molecules and SDH. Consistent with the virtual verification results of a molecular docking comparison, the in vitro antifungal bioassays indicated that the skeleton structure of title compounds should be optimized as an N'-(4-phenoxyphenyl)-1H-pyrazole-4-carbohydrazide scaffold. Strikingly, N'-(4-phenoxyphenyl)-1H-pyrazole-4-carbohydrazide derivatives 11o against Rhizoctonia solani, 11m against Fusarium graminearum, and 11g against Botrytis cinerea exhibited excellent antifungal effects, with corresponding EC50 values of 0.14, 0.27, and 0.52 µg/mL, which were obviously better than carbendazim against R. solani (0.34 µg/mL) and F. graminearum (0.57 µg/mL) as well as penthiopyrad against B. cinerea (0.83 µg/mL). The relative studies on an in vivo bioassay against R. solani, bioactive evaluation against SDH, and molecular docking were further explored to ascertain the practical value of compound 11o as a potential fungicide targeting SDH. The present work provided a non-negligible complement for the structural optimization of antifungal leads targeting SDH.

Mechanism of Action of Novel Pyrazole Carboxamide Containing a Diarylamine Scaffold against Rhizoctonia solani.

In the last few decades, Rhizoctonia solani causing rice sheath blight has resulted in a lot of economic losses in the world. Therefore, many novel pyrazole carboxamide fungicides have been intensively researched and employed to fight against it. In this regard, in recent years, our group reported a novel pyrazole carboxamide containing a diarylamine scaffold with good antifungal activity against rice sheath blight in the pot test and field trial. Following this project, the antifungal mechanism of action of the pyrazole carboxamide has been elucidated in this work. The antifungal result showed that compound SCU2028, N-[2-[(3-chlorophenyl)amino]-phenyl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide, was equivalent to the commercial fungicide thifluzamide and its EC50 value was 0.022 mg/L against R. solani. Also, the observation results by scanning electron microscopy and transmission electron microscopy showed that it could destroy the fungus' cell walls or membranes and result in the leakage of contents and increase of the number of mitochondria and abnormal morphology. Meanwhile, the result on the mitochondrial membrane potential (MMP) showed that it could decrease R. solani's MMP. Furthermore, the results by label-free quantitative proteomic analysis showed that 1153 proteins were found after R. solani was treated with compound SCU2028, including 212 proteins in the control group and 257 proteins in the treatment group. A total of 142 differential proteins were obtained, of which 92 proteins were upregulated and 50 proteins were downregulated. The differentially expressed proteins affected a series of physiological and biochemical pathways in the mitochondria, endoplasmic reticulum, ribosome, and other related GO and KEGG pathways. In particular, the inhibition of the respiratory chain caused by the TCA cycle and oxidative phosphorylation KEGG pathway indicated that complex II (succinate dehydrogenase) and complex IV (cytochrome oxidase) might be compound SCU2028's main action targets. In addition, multiple experiments of qRT-PCR, enzyme activity detection, and molecular docking confirmed complex II and complex IV as targets. It could be seen that these findings provided a theoretical support for further research and development of the pyrazole carboxamide fungicides.

Global Characterization of GH10 Family Xylanase Genes in Rhizoctonia cerealis and Functional Analysis of Xylanase RcXYN1 During Fungus Infection in Wheat.

Wheat (Triticum aestivum L.) is an important staple crop. Rhizoctonia cerealis is the causal agent of diseases that are devastating to cereal crops, including wheat. Xylanases play an important role in pathogenic infection, but little is known about xylanases in R. cerealis. Herein, we identified nine xylanase-encoding genes from the R. cerealis genome, named RcXYN1-RcXYN9, examined their expression patterns, and investigated the pathogenicity role of RcXYN1. RcXYN1-RcXYN9 proteins contain two conserved glutamate residues within the active motif in the glycoside hydrolase 10 (GH10) domain. Of them, RcXYN1-RcXYN4 are predicted to be secreted proteins. RcXYN1-RcXYN9 displayed different expression patterns during the infection process of wheat, and RcXYN1, RcXYN2, RcXYN5, and RcXYN9 were expressed highly across all the tested inoculation points. Functional dissection indicated that the RcXYN1 protein was able to induce necrosis/cell-death and H2O2 generation when infiltrated into wheat and Nicotiana benthamiana leaves. Furthermore, application of RcXYN1 protein followed by R. cerealis led to significantly higher levels of the disease in wheat leaves than application of the fungus alone. These results demonstrate that RcXYN1 acts as a pathogenicity factor during R. cerealis infection in wheat. This is the first investigation of xylanase genes in R. cerealis, providing novel insights into the pathogenesis mechanisms of R. cerealis.

Antifungal Action of Antifungalmycin N2 Against Rhizoctonia solani by Disrupting Cell Membrane and Inhibiting Succinate Dehydrogenase.

Antifungalmycin N2 (3-methyl-3,5-amino-4-vinyl-2-pyrone, C6H7O2N) was a novel structural antifungal metabolite produced by Streptomyces sp. strain N2. Our previous study reported that the antagonistic interaction between antifungalmycin N2 and Rhizoctonia solani was accompanied by an oxidative stress in R. solani cell, indicating a probable damage occurred in the cell membranes and mitochondria. To verify this, the present study focused on investigating the effects of antifungalmycin N2 on the structure and function of cell membranes and mitochondria of R. solani. Morphological observations in transmission electron microscopy and fluorescence microscope showed that cell membranes of R. solani were damaged, and its cytoplasmic organelles were disorganized when treated with antifungalmycin N2. Meanwhile, the kinetics of membrane-related physiological and biochemical parameters, such as the increased malondialdehyde level, dropped ergosterol formation, and enhanced electrical conductivity in R. solani mycelia, further confirmed that antifungalmycin N2 would disrupt the cell membrane structure and function. More significantly, antifungalmycin N2 had a significantly inhibitory effect on the succinate dehydrogenase (SDH) activity of R. solani, and indicated that the mode and site of action of antifungalmycin N2 against R. solani might be similar to the existing succinate dehydrogenase inhibitors fungicides by binding in the ubiquinone-binding site. In conclusion, the above results demonstrated that the mode and site of action of antifungalmycin N2 targeted to cell membrane and SDH of R. solani, thus exerting the antifungal activity by damaging cell membrane structure and function, together with inhibiting the SDH activity.

Synthesis and Biological Activity of Novel Succinate Dehydrogenase Inhibitor Derivatives as Potent Fungicide Candidates.

In searching for novel fungicidal leads, the novel bioactive succinate dehydrogenase inhibitor (SDHI) derivatives were designed and synthesized by the inversion of carbonyl and amide groups. Bioassay indicated that compound 5i stood out with a broad spectrum of in vitro activity against five fungi. Its EC50 value (0.73 µg/mL) was comparable to that of boscalid (EC50 of 0.51 µg/mL) and fluxapyroxad (EC50 of 0.19 µg/mL) against Sclerotinia sclerotiorum. For Rhizoctonia cerealis, 5i and 5p with EC50 values of 4.61 and 6.48 µg/mL, respectively, showed significantly higher activity than fluxapyroxad with the EC50 value of 16.99 µg/mL. In vivo fungicidal activity of 5i exhibited an excellent inhibitory rate (100%) against Puccinia sorghi at 50 µg/mL, while the positive control boscalid showed only a 70% inhibitory rate. Moreover, 5i showed promising fungicidal activity with a 60% inhibitory rate against Rhizoctonia solani at 1 µg/mL, which was better than that of boscalid (30%). Compound 5i possessed better in vivo efficacy against P. sorghi and R. solani than boscalid. Molecular docking showed that even the carbonyl oxygen atom of 5i was far from the pyrazole ring. It could also form hydrogen bonds toward the hydroxyl hydrogen and amino hydrogen of TYR58 and TRP173 on SDH, respectively, which consisted of the positive control fluxapyroxad. Fluorescence quenching analysis and SDH enzymatic inhibition studies also validated its mode of action. Our studies showed that 5i was worthy of further investigation as a promising fungicide candidate.

Discovery of N-(4-fluoro-2-(phenylamino)phenyl)-pyrazole-4-carboxamides as potential succinate dehydrogenase inhibitors.

Succinate dehydrogenase (SDH), an essential component of cellular respiratory chain and tricarboxylic acid (or Krebs) cycle, has been identified as one of the most significant targets for pharmaceutical and agrochemical. Herein, with the aim of discovery of new antifungal lead structures, a class of novel N-(4-fluoro-2-(phenylamino)phenyl)-pyrazole-4-carboxamides were designed, synthesized and evaluated for their biological activities. They were bioassayed against seven phytopathogenic fungi, Rhizoctonia solani, Phytophthora infestans, Fusarium oxysporum f. sp. vasinfectum, Botryosphaeria dothidea, Gibberella zeae, Alternaria alternate and Fusarium oxysporum f. sp. niveum. The results indicated that most of the compounds displayed good antifungal activities, especially against R. solani. Among them, compounds 7 and 12 exhibited higher antifungal activities against R. solani in vitro with EC50 value of 0.034 mg/L and 0.021 mg/L, being superior to the commercially available fungicide bixafen (EC50 = 0.043 mg/L). Pot tests against R. solani showed that in vivo EC50 values of compounds 7 (2.694 mg/L) and 12 (2.331 mg/L) were higher than that of bixafen (3.724 mg/L). In addition, inhibitory activity of compound 12 against SDH indicated compound 12 (IC50 = 1.836 mg/L) showed good inhibitory activity against SDH, being close to bixafen's inhibitory activity (IC50 = 1.222 mg/L). And, molecular modeling of the SDH-compound 12 complex suggested that compound 12 could strongly bind to and interact with the binding site of the SDH. The results of the present work showed that N-(4-fluoro-2-(phenylamino)phenyl)-pyrazole-4-carboxamides were a new fungicides for discovery of SDH inhibitors and worth further study.

Distinct Regioselectivity of Fungal P450 Enzymes for Steroidal Hydroxylation.

In this study, we identified two P450 enzymes (CYP5150AP3 and CYP5150AN1) from Thanatephorus cucumeris NBRC 6298 by combination of transcriptome sequencing and heterologous expression in Pichia pastoris The biotransformation of 11-deoxycortisol and testosterone by Pichia pastoris whole cells coexpressing the cyp5150ap3 and por genes demonstrated that the CYP5150AP3 enzyme possessed steroidal 7ß-hydroxylase activities toward these substrates, and the regioselectivity was dependent on the structures of steroidal compounds. CYP5150AN1 catalyzed the 2ß-hydroxylation of 11-deoxycortisol. It is interesting that they display different regioselectivity of hydroxylation from that of their isoenzyme, CYP5150AP2, which possesses 19- and 11ß-hydroxylase activities.IMPORTANCE The steroidal hydroxylases CYP5150AP3 and CYP5150AN1 together with the previously characterized CYP5150AP2 belong to the CYP5150A family of P450 enzymes with high amino acid sequence identity, but they showed completely different regioselectivities toward 11-deoxycortisol, suggesting the regioselectivity diversity of steroidal hydroxylases of CYP5150 family. They are also distinct from the known bacterial and fungal steroidal hydroxylases in substrate specificity and regioselectivity. Biocatalytic hydroxylation is one of the important transformations for the functionalization of steroid nucleus rings but remains a very challenging task in organic synthesis. These hydroxylases are useful additions to the toolbox of hydroxylase enzymes for the functionalization of steroids at various positions.

Hydrolysis of chlorogenic acid in apple juice using a p-coumaryl esterase of Rhizoctonia solani.

BACKGROUND: Apple juice is rich in polyphenolic compounds, especially in chlorogenic acid. A sour and bitter taste has been attributed to the compound. Chlorogenic acid in coffee powder was quickly hydrolysed by a p-coumaryl esterase of Rhizoctonia solani (RspCAE) at its optimal pH of 6.0. It was unknown, however, if RspCAE would also degrade chlorogenic acid under the strongly acidic conditions (pH 3.3) present in apple juice. RESULTS: Treatment of apple juice with RspCAE led to a chlorogenic acid degradation from 53.38 ± 0.94 mg L-1 to 21.02 ± 1.47 mg L-1 . Simultaneously, the caffeic acid content increased from 6.72 ± 0.69 mg L-1 to 19.33 ± 1.86 mg/L-1 . The aroma profile of the enzymatically treated sample and a control sample differed in only one volatile. Vitispirane had a higher flavour dilution factor in the treated juice. Sensory analysis showed no significant difference in the taste profile ( p < 0.05). CONCLUSION: These results demonstrated a high stability and substrate specificity of RspCAE. An increase in caffeic acid and a concurrent decrease in chlorogenic acid concentration may exert a beneficial effect on human health. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The peculiar physiological responses of Rhizoctonia solani under the antagonistic interaction coupled by a novel antifungalmycin N2 from Streptomyces sp. N2.

A novel antifungalmycin N2 (3-methyl-3,5-amino-4-vinyl-2-pyrone, C6H7O2N) was previously discovered from Streptomyces sp. N2, which exerted a broad-spectrum antagonistic activity against phytopathogenic fungi. To provide comprehensive insights into the antagonistic mechanisms and biocontrol efficacy of antifungalmycin N2, the present work investigated the physiological responses of Rhizoctonia solani under interaction with antifungalmycin N2. First, the mycelial growth of R. solani was significantly inhibited by antifungalmycin N2 during liquid shake-flask culture. Morphological observations showed that the morphogenesis of R. solani was influenced by antifungalmycin N2, in which the hyphae became severely shriveled and flattened, irregularly folded and branched. Additionally, an obvious accumulation of reactive oxygen species (ROS) was detected in R. solani hyphae, indicating oxidative stress induced by antifungalmycin N2. Further results showed that chitinase activity and its hydrolytic N-acetylglucosamine were significantly accelerated by antifungalmycin N2, demonstrating the cell wall of R. solani was damaged. Interestingly, the enzymatic antioxidant activities of R. solani were significantly induced in response to a relatively low concentration of antifungalmycin N2 (1.44-5.77 µg/mL). However, all antioxidant enzymes became highly inactive when the antifungalmycin N2 was increased to 11.53 µg/mL, suggesting that the enzymatic antioxidant system in R. solani was probably collapsed by the oxidative stress beyond its acceptance scope. In conclusion, antifungalmycin N2 exerted its antagonistic activity by inducing both cell wall degradation and oxidative stress in R. solani, thus leading to fungal morphogenesis and autolysis. Meanwhile, R. solani could induce and activate its antioxidant enzymes as a defence response to the oxidative stress caused by antifungalmycin N2.

Discovery of Novel Thiazole Carboxamides as Antifungal Succinate Dehydrogenase Inhibitors.

To contribute molecular diversity for novel fungicide development, a series of novel thiazole carboxamides were rationally designed, synthesized, and characterized with the succinate dehydrogenase (SDH) as target. Bioassay indicated that compound 6g showed the similar excellent SDH inhibition as that of Thifluzamide with IC50 of 0.56 mg/L and 0.55 mg/L, respectively. Some derivatives displayed improved in vitro fungicidal activities against Rhizoctonia cerealis and Sclerotinia sclerotiorum with EC50 of 1.2-16.4 mg/L and 0.5-1.9 mg/L. Surprisingly, 6g showed promising in vitro fungicidal activities against R. cerealis and S. sclerotiorum with EC50 of 6.2 and 0.6 mg/L, respectively, which was superior to Thifluzamide with the EC50 of 22.1 and 4.4 mg/L, respectively. Additionally, compounds 6c and 6g displayed excellent in vivo fungicidal activities against S. sclerotiorum on Brassica napus L. leaves with protective activity of 75.4% and 67.3% at 2.0 mg/L, respectively, while Thifluzamide without activity at 5.0 mg/L. Transcriptomic analysis of S. sclerotiorum treated with 6g by RNA sequencing indicated the down-regulation of succinate dehydrogenase gene SDHA and SDHB, and the inhibition of the TCA-cycle.

Effects and inhibition mechanism of phenazine-1-carboxamide on the mycelial morphology and ultrastructure of Rhizoctonia solani.

The purpose of this research was to explore the effect of phenazine-1-carboxamide (PCN) on Rhizoctonia solani and to elucidate its mechanisms of action. The toxicity of PCN to R. solani was measured using a growth rate method. The results indicated that PCN inhibited R. solani with a 50% effective concentration (EC50) of 9.0934µg/mL. The mycelia of R. solani were then exposed to 18.18µg/mL (2EC50) of PCN. Optical microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) were used to observe the effects of PCN on mycelial morphology and ultrastructure. Following the PCN treatment, the optical microscopy observations revealed that the mycelia appeared twisted; the branching mycelia grew, but the main mycelia did not grow following branching; and the mycelial roots possessed more vacuoles. SEM observations revealed that the mycelia were locally swollen and exhibited a sharp decrease in prominence. TEM observations showed that the cell wall became thin and deformed; the mitochondria disappeared; the septum twisted; and most of the organelles were difficult to discern. Conversely, all of the organelles could be clearly observed in the control. We then used real-time quantitative PCR and an enzyme activity testing kit to further explore the effects of PCN on the cell wall and mitochondria. Physiological and biochemical results demonstrated that both the cell wall and mitochondria constitute are PCN targets. PCN inhibited the activities of chitin synthetase and complex I of the mitochondria electron transport chain. Molecular experiments demonstrated that PCN controlled the growth of R. solani mycelia by inhibiting the expression level of chitin synthetase genes. Future research on PCN should investigate its influence on metabolic pathways, thereby aiding in the potential development of novel pesticides.

Enzymatic mitigation of 5-O-chlorogenic acid for an improved digestibility of coffee.

A p-coumaroyl esterase from Rhizoctonia solani was used to decrease 5-O-chlorogenic acid (5-CQA) content in coffee powder. HPLC-UV showed a decline of up to 98% of 5-CQA after the enzyme treatment. Effects on aroma were determined by means of aroma extract dilution analysis. Flavour dilution factors of treated and control extract differed in four volatile compounds only. Effect on aroma and taste was evaluated by sensory tests. No significant differences were perceived, and no off-flavour nor off-taste was noted. As chlorogenic acids are suspected to cause stomach irritating effects in sensitive people, the enzyme treatment offers a technically feasible approach to improve the quality of coffee beverages by reducing 5-CQA concentration without significantly affecting the aroma and taste profile.

Activation of Recombinantly Expressed l-Amino Acid Oxidase from Rhizoctonia solani by Sodium Dodecyl Sulfate.

l-Amino acid oxidases (l-AAO) catalyze the oxidative deamination of l-amino acids to the corresponding α-keto acids. The non-covalently bound cofactor FAD is reoxidized by oxygen under formation of hydrogen peroxide. We expressed an active l-AAO from the fungus Rhizoctonia solani as a fusion protein in E. coli. Treatment with small amounts of the detergent sodium dodecyl sulfate (SDS) stimulated the activity of the enzyme strongly. Here, we investigated whether other detergents and amphiphilic molecules activate 9His-rsLAAO1. We found that 9His-rsLAAO1 was also activated by sodium tetradecyl sulfate. Other detergents and fatty acids were not effective. Moreover, effects of SDS on the oligomerization state and the protein structure were analyzed. Native and SDS-activated 9His-rsLAAO1 behaved as dimers by size-exclusion chromatography. SDS treatment induced an increase in hydrodynamic radius as observed by size-exclusion chromatography and dynamic light scattering. The activated enzyme showed accelerated thermal inactivation and an exposure of additional protease sites. Changes in tryptophan fluorescence point to a more hydrophilic environment. Moreover, FAD fluorescence increased and a lower concentration of sulfites was sufficient to form adducts with FAD. Taken together, these data point towards a more open conformation of SDS-activated l-amino acid oxidase facilitating access to the active site.

Synthesis of novel fenfuram-diarylether hybrids as potent succinate dehydrogenase inhibitors.

Twelve novel fenfuram-diarylether hybrids were designed, synthesized and characterized by 1H NMR and MS. Their in vitro antifungal activities were evaluated against five phytopathogenic fungi by mycelial growth inhibition method. Most compounds showed significant antifungal effect on Rhizoctonia solani and Sclerotinia sclerotiorum. Compound 1c exhibited the most potent antifungal effect on R. solani with an EC50 value of 0.242mg/L, superior to the commercial fungicide boscalid (EC50=1.758mg/L) and the lead fungicide fenfuram (EC50=7.691mg/L). Molecular docking revealed that compound 1c featured a higher affinity for succinate dehydrogenase (SDH) than fenfuram. Furthermore, it was shown that the 2-chlorophenyl group of compound 1c formed a π-π stacking with D/Tyr-128 and a Cl-π interaction with B/His-249, which made compound 1c more active than fenfuram against SDH.

Discovery of Novel Succinate Dehydrogenase Inhibitors by the Integration of in Silico Library Design and Pharmacophore Mapping.

Succinate dehydrogenase (SDH) has been demonstrated as a promising target for fungicide discovery. Crystal structure data have indicated that the carboxyl "core" of current SDH inhibitors contributed largely to their binding affinity. Thus, identifying novel carboxyl "core" SDH inhibitors would remarkably improve the biological potency of current SDHI fungicides. Herein, we report the discovery and optimization of novel carboxyl scaffold SDH inhibitor via the integration of in silico library design and a highly specific amide feature-based pharmacophore model. To our delight, a promising SDH inhibitor, A16c (IC50 = 1.07 µM), with a novel pyrazol-benzoic scaffold was identified, which displayed excellent activity against Rhizoctonia solani (EC50 = 11.0 µM) and improved potency against Sclerotinia sclerotiorum (EC50 = 5.5 µM) and Phyricularia grisea (EC50 = 12.0 µM) in comparison with the positive control thifluzamide, with EC50 values of 0.09, 33.2, and 33.4 µM, respectively. The results showed that our virtual screening strategy could serve as a powerful tool to accelerate the discovery of novel SDH inhibitors.

A p-coumaroyl esterase from Rhizoctonia solani with a pronounced chlorogenic acid esterase activity.

Extracellular esterase activity was detected in submerged cultures of Rhizoctonia solani grown in the presence of sugar beet pectin or Tween 80. Putative type B feruloyl esterase (FAE) coding sequences found in the genome data of the basidiomycete were heterologously expressed in Pichia pastoris. Recombinant enzyme production on the 5-L bioreactor scale (Rs pCAE: 3245UL-1) exceeded the productivity of the wild type strain by a factor of 800. Based on substrate specificity profiling, the purified recombinant Rs pCAE was classified as a p-coumaroyl esterase (pCAE) with a pronounced chlorogenic acid esterase side activity. The Rs pCAE was also active on methyl cinnamate, caffeate and ferulate and on feruloylated saccharides. The unprecedented substrate profile of Rs pCAE together with the lack of sequence similarity to known FAEs or pCAEs suggested that the Rs pCAE represents a new type of enzyme. Hydroxycinnamic acids were released from agro-industrial side-streams, such as destarched wheat bran (DSWB), sugar beet pectin (SBP) and coffee pulp (CP). Overnight incubation of coffee pulp with the Rs pCAE resulted in the efficient release of p-coumaric (100%), caffeic (100%) and ferulic acid (85%) indicating possible applications for the valorization of food processing wastes and for the enhanced degradation of lignified biomass.

Structure-Based Discovery of Potential Fungicides as Succinate Ubiquinone Oxidoreductase Inhibitors.

A series of diphenyl ether-containing pyrazole-carboxamide derivatives was designed and synthesized as new succinate ubiquinone oxidoreductase (SQR) inhibitors. This highly potent molecular scaffold was developed from a moderately activie hit 3, obtained from our previous pharmacophore-linked fragment virtual screening (PFVS) method. The results of greenhouse tests indicated that some analogues showed good SQR inhibitory activity, with promising fungicidal activity against Rhizoctonia solani and Sphaerotheca fuliginea at a dosage of 200 mg/L. Most surprisingly, compound 62 showed the highest SQR inhibitory activity with a Ki value of 0.081 µM, about 4-fold more potent than penthiopyrad (Ki = 0.307 µM). In addition, compounds 43 and 62 displayed excellent fungicidal activity even at a dosage as low as 6.25 mg/L, which was superior to thifluzamide. Moreover, compound 62 exhibited excellent protection effect against R. solani and provided about 81.2% protective control efficancy after 21 days with two sprayings. The present work indicated that these two compounds could be used as potential agricultural fungicides targeting SQR.

Homozygous and heterozygous point mutations in succinate dehydrogenase subunits b, c and d of Rhizoctonia cerealis conferring resistance to thifluzamide.

BACKGROUND: Thifluzamide, a succinate dehydrogenase inhibitor (SDHI) fungicide, is a promising fungicide for controlling wheat sharp eyespot (WSE). WSE is caused by Rhizoctonia cerealis. Information on the resistance mechanism of this pathogen to thifluzamide remains unavailable. RESULTS: We used selective reculturing and UV mutagenesis to generate thifluzamide-resistant mutants. Thifluzamide-resistant mutants were only generated through UV mutagenesis. Sequence analysis of succinate dehydrogenase (Sdh) genes revealed that two mutants had no mutation in RCSdhB, RCSdhC and RCSdhD, and the other 18 mutants all had at least one mutation in RCSdhB, RCSdhC or RCSdhD, either in a homozygous or heterozygous state. The majority of mutants included either RCSdhD-H116Y or RCSdhC-H139Y. They showed slight resistance to boscalid, bixafen and penflufen. Only one mutant possessed RCSdhB-H246Y, and it showed medium resistance to boscalid and penflufen and a slight resistance to bixafen. All the thifluzamide mutants were sensitive to flutolanil. Compared with their parental isolates, these mutants present no or minor fitness penalties. CONCLUSION: Homozygous and heterozygous point mutations in the succinate dehydrogenase subunits b, c and d of R. cerealis may be involved in thifluzamide resistance. © 2016 Society of Chemical Industry.

Recombinant expression and characterization of a L-amino acid oxidase from the fungus Rhizoctonia solani.

L-Amino acid oxidases (L-AAOs) catalyze the oxidative deamination of L-amino acids to the corresponding α-keto acids, ammonia, and hydrogen peroxide. L-AAOs are homodimeric enzymes with FAD as a non-covalently bound cofactor. They are of potential interest for biotechnological applications. However, heterologous expression has not succeeded in producing large quantities of active recombinant L-AAOs with a broad substrate spectrum so far. Here, we report the heterologous expression of an active L-AAO from the fungus Rhizoctonia solani in Escherichia coli as a fusion protein with maltose-binding protein (MBP) as a solubility tag. After purification, it was possible to remove the MBP-tag proteolytically without influencing the enzyme activity. MBP-rsLAAO1 and 9His-rsLAAO1 converted basic and large hydrophobic L-amino acids as well as methyl esters of these L-amino acids. The progress of the conversion of L-phenylalanine and L-leucine into the corresponding α-keto acids was determined by HPLC and 1H-NMR analysis of reaction mixtures, respectively. Enzymatic activity was stimulated 50-100-fold by SDS treatment. K m values ranging from 0.9-10 mM and v max values from 3 to 10 U mg-1 were determined after SDS activation of 9His-rsLAAO1 for the best substrates. The enzyme displayed a broad pH optimum between pH 7.0 and 9.5. In summary, a successful overexpression of recombinant L-AAO in E. coli was established that results in a promising enzymatic activity and a broad substrate spectrum for biotechnological application.