Improvement of Heterologous Soluble Expression of L-amino Acid Oxidase Using Logistic Regression.

Successful implementation of enzymes in practical application hinges on the development of efficient mass production techniques. However, in a heterologous expression system, the protein is often unable to fold correctly and, thus, forms inclusion bodies, resulting in the loss of its original activity. In this study, we present a new and more accurate model for predicting amino acids associated with an increased L-amino acid oxidase (LAO) solubility. Expressing LAO from Rhizoctonia solani in Escherichia coli and combining random mutagenesis and statistical logistic regression, we modified 108 amino acid residues by substituting hydrophobic amino acids with serine and hydrophilic amino acids with alanine. Our results indicated that specific mutations in Euclidean distance, glycine, methionine, and secondary structure increased LAO expression. Furthermore, repeated mutations were performed for LAO based on logistic regression models. The mutated LAO displayed a significantly increased solubility, with the 6-point and 58-point mutants showing a 2.64- and 4.22-fold increase, respectively, compared with WT-LAO. Ultimately, using recombinant LAO in the biotransformation of α-keto acids indicates its great potential as a biocatalyst in industrial production.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

Genome analysis of the sugar beet pathogen Rhizoctonia solani AG2-2IIIB revealed high numbers in secreted proteins and cell wall degrading enzymes.

BACKGROUND: Sugar beet (Beta vulgaris) is a crop cultivated for its high content in sugar, but it is vulnerable to many soil-borne pathogens. One of them is the basidiomycete Rhizoctonia solani. This fungal species has a compatibility system regulating hyphal fusions (anastomosis). Consequently, R. solani species are categorized in anastomosis groups (AGs). AG2-2IIIB isolates are most aggressive on sugar beet. In the present study, we report on the draft genome of R. solani AG2-2IIIB using the Illumina technology. Genome analysis, interpretation and comparative genomics of five sequenced R. solani isolates were carried out. RESULTS: The draft genome of R. solani AG2-2IIIB has an estimated size of 56.02 Mb. In addition, two normalized EST libraries were sequenced. In total 20,790 of 21,980 AG2-2IIIB isotigs (transcript isoforms) were mapped on the genome with more than 95 % sequence identity. The genome of R. solani AG2-2IIIB was predicted to harbor 11,897 genes and 4908 were found to be isolate-specific. R. solani AG2-2IIIB was predicted to contain 1142 putatively secreted proteins and 473 of them were found to be unique for this isolate. The R. solani AG2-2IIIB genome encodes a high number of carbohydrate active enzymes. The highest numbers were observed for the polysaccharide lyases family 1 (PL-1), glycoside hydrolase family 43 (GH-43) and carbohydrate estarase family 12 (CE-12). Transcription analysis of selected genes representing different enzyme clades revealed a mixed pattern of up- and down-regulation six days after infection on sugar beets featuring variable levels of resistance compared to mycelia of the fungus grown in vitro. CONCLUSIONS: The established R. solani AG2-2IIIB genome and EST sequences provide important information on the gene content, gene structure and transcriptional activity for this sugar beet pathogen. The enriched genomic platform provides an important platform to enhance our understanding of R. solani biology.

Suppression subtractive hybridization and comparative expression of a pore-forming toxin and glycosyl hydrolase genes in Rhizoctonia solani during potato sprout infection.

Rhizoctonia solani is a plant pathogenic fungus that causes black scurf on tubers and stem and stolon canker on underground parts of potato plant. Early in the season, the fungus attacks germinating sprouts underground before they emerge from the soil. Damage at this stage results in delayed emergence of weakened plants with poor and uneven stands. The mechanism underlying this phenomenon has been investigated in this study by coupling a cDNA-suppression subtractive hybridization (SSH) library to differential screening to identify transcripts of R. solani that are down-regulated during infection of potato sprouts. We report on the identification of 33 unique genes with functions related to carbohydrate binding, vitamin synthesis, pathogenicity, translation, ATP and nucleic acid binding and other categories. RACE-PCR was used to clone and characterize the first full-length cDNA clones, RSENDO1 and RSGLYC1 that encode for an eukaryotic delta-endotoxin CytB protein and an intracellular glycosyl hydrolase, respectively. Quantitative real-time PCR revealed the down-regulation of RSENDO1 during infection of potato sprouts and the up-regulation of RSGLYC1 when the fungus was grown on a cellulose-based nutrient medium. In contrast, additional experiments have highlighted the down-regulation of RSENDO1 when R. solani was co-cultured with the mycoparasite Stachybotrys elegans and the bacterial antagonist Bacillus subtilis B26. These results advance our understanding of R. solani-potato interaction in subterranean parts of the plant. Such approaches could be considered in building an efficient integrated potato disease management program.

Antifungal activity of various essential oils against Rhizoctonia solani and Macrophomina phaseolina as major bean pathogens.

AIMS: The main objective of this study was to investigate the effect of various essential oils (EOs) to decrease the activity of cell wall degrading enzymes (CWDEs) produced by fungal phytopathogens, which are associated with disease progress. Also, effect of seed treatment and foliar application of peppermint EO and its main constituent, menthol, on diseases caused by two necrotrophic pathogens on bean was investigated. METHODS AND RESULTS: Antifungal activity of EOs on Rhizoctonia solani and Macrophomina phaseolina, as bean pathogens, was evaluated. The EOs of Mentha piperita, Bunium persicum and Thymus vulgaris revealed the highest antifungal activity against fungi. The EO of M. piperita had the lowest minimum inhibitory concentration (MIC) for R. solani among the three EOs tested. This pathogen did not grow in the presence of M. piperita, B. persicum and T. vulgaris EOs at 850, 1200 and 1100 ppm concentrations, respectively. The B. persicum EO had the lowest MIC for M. phaseolina as this fungus did not grow in the presence of M. piperita, B. persicum and T. vulgaris EOs at concentrations of 975, 950 and 1150 ppm, respectively. Hyphae exposed to EOs showed structural changes. Activities of cellulase and pectinase, as main CWDEs of pathogens, decreased by EOs at low concentration without effect on fungal growth. Seed treatment and foliar application of peppermint EO and/or menthol significantly reduced the development of bean diseases caused by both fungi. Higher capability of menthol than peppermint EO in decreasing diseases on bean was observed. CONCLUSIONS: Reducing CDWEs activity is a mechanism of EOs' effect on fungi. Higher antifungal activity of menthol compared to peppermint EO was observed not only in vitro but also in vivo. SIGNIFICANCE AND IMPACT OF THE STUDY: Effect of EOs on CWDEs involved in pathogenesis is described in this study for the first time. Menthol can be used as a botanical fungicide to control destructive fungal diseases on bean.

Baseline sensitivity and resistance risk assessmemt of Rhizoctonia cerealis to thifluzamide, a succinate dehydrogenase inhibitor.

During 2010-2012, a total of 120 isolates of Rhizoctonia cerealis were collected from wheat with symptoms of sharp eyespot in four provinces (Henan, Shandong, Anhui and Jiangsu) in China. All the isolates were determined for baseline sensitivity to thifluzamide, a succinate dehydrogenase inhibitor (SDHI) with strong antifungal activity. The sampled pathogenic populations, never exposed to SDHIs, had similar sensitivity to trifluzamide (0.025-0.359 µg/ml) in the four regions and over the two years. The baseline sensitivity was distributed as a skewed unimodal curve with a mean EC50 value (effective concentrations for 50% inhibiting mycelial growth) of 0.064 ± 0.013 µg/ml. The resistance risk of R. cerealis to thifluzamide was further evaluated in vitro. Two thifluzamide-resistant mutants of R. cerealis were obtained by culturing on thifluzamide-amended plates. The resistance factors (RF = EC50 value of a mutant/EC50 value of the wild type progenitor of the mutant) were 120 and 40 for two R. cerealis mutants, respectively. All the mutants exhibited similar fitness after 10 successive transfers when compared to their wild-type parents in mycelial growth, sclerotia production, and virulence. However, the two thifluzamide-resistant mutants differed significantly in sensitivity to boscalid and flutolanil. Therefore, a low-to-moderate risk of resistance development was recommended for thifluzamide.

[Cloning, prokaryotic expression and bioinformatics of Rspg1 gene of Rhizoctonia solani].

OBJECTIVE: The study was aimed at understanding the roles of polygalacturonases in the pathogenicity and the interaction between Rhizoctonia solani and rice. METHODS: According to the sequences of Rspg1 of R. solani deposited in GenBank, a pair of specific primers was designed. The gene Rspg1 was cloned and expressed using prokaryotic expression tool to elucidate its biological characteristics. The structures of the protein RsPG1 were predicted using bioinformatics tools. RESULTS: A 1395-bp fragment including an open reading frame (OFR) of Rspg1 was amplified from the genomic DNA of the pathogen. Compared with RT-PCR results, it was found that this sequence fragment contains five introns (positions 278-334, 545-601, 657-715, 1090-1155 and 1244-1304) and one 1095 bp ORF. The ORF was predicted to encode 364 amino acids. Bioinformatics analysis showed that RsPG1 contains an 18-amino acid signal peptide and 4 conserved sequence segments (180NTD, 202DD, 223GHG and 255RIK) characteristic of all the polygalacturonases. The main structural elements of the secondary structure are alpha-helix, beta-sheet and random coil. Six cysteines form three disulfide bonds (Cys24-Cys40, Cys204-Cys220 and Cys329-Cys333). Transmembrane prediction analysis suggested that RsPG1 could be secreted outside the cell. Tertiary structure is a right-handed helix which consisted of ten repeated beta-sheet, forming an opening activity cleft. CONCLUSION: RsPG1 is tentatively a 40 kDa protein with polygalacturonase enzyme activity at 277.78 U/mg. It is probably a secreted protein and has characteristics of all the polygalacturonases. The results can help to further understand the roles that R. solani polygalacturonases play during the pathogenicity and how the pathogen interacts with the host.

Design, Synthesis, and Antifungal Activities of Novel Potent Fluoroalkenyl Succinate Dehydrogenase Inhibitors.

The development of new fungicide molecules is a crucial task for agricultural chemists to enhance the effectiveness of fungicides in agricultural production. In this study, a series of novel fluoroalkenyl modified succinate dehydrogenase inhibitors were synthesized and evaluated for their antifungal activities against eight fungi. The results from the in vitro antifungal assay demonstrated that compound 34 exhibited superior activity against Rhizoctonia solani with an EC50 value of 0.04 µM, outperforming commercial fluxapyroxad (EC50 = 0.18 µM) and boscalid (EC50 = 3.07 µM). Furthermore, compound 34 showed similar effects to fluxapyroxad on other pathogenic fungi such as Sclerotinia sclerotiorum (EC50 = 1.13 µM), Monilinia fructicola (EC50 = 1.61 µM), Botrytis cinerea (EC50 = 1.21 µM), and also demonstrated protective and curative efficacies in vivo on rapeseed leaves and tomato fruits. Enzyme activity experiments and protein-ligand interaction analysis by surface plasmon resonance revealed that compound 34 had a stronger inhibitory effect on succinate dehydrogenase compared to fluxapyroxad. Additionally, molecular docking and DFT calculation confirmed that the fluoroalkenyl unit in compound 34 could enhance its binding capacity with the target protein through p-π conjugation and hydrogen bond interactions.

[Secretion of proteolytic enzymes by three phytopathogenic microorganisms].

Serine proteinases from three phytopathogenic microorganisms that belong to different fungal families and cause diseases in potatoes were studied and characterized. The oomycete Phytophthora infestans (Mont.) de Bary and the fungi Rhizoctonia solani and Fusarium culmorum were shown to secrete serine proteinases. An analysis of the substrate specificity of these enzymes and their sensitivity to synthetic and protein inhibitors allowed us to refer them to trypsin- and subtilisin-like proteinases. The correlation between the trypsin- and subtilisin-like proteinases depended on the composition of the culture medium, particularly on the form of the nitrogen source. A phylogenetic analysis was carried out. In contrast to basidiomycetes R. solani, ascomycetes F. culmorum and oomycetes P. infestans produced a similar set of exoproteinases, although they had more distant phylogenetic positions. This indicated that the secretion of serine proteinases by various phytopathogenic microorganisms also depended on their phylogenetic position. These results allowed us to suggest that exoproteinases from phytopathogenic fungi play a different role in pathogenesis. They may promote the adaptation of fungi if the range of hosts is enlarged. On the other hand, they may play an important role in the survival of microorganisms in hostile environements outside their hosts.

Increase in extracellular inulinase production for a new Rhizoctonia ssp. strain by using buckwheat (Fagopyrum esculentum) flour as a single carbon source.

AIMS: A newly isolated strain of Rhizoctonia ssp. was used for the production of extracellular inulinase. Previously, the qualitative effects of some carbon and nitrogen sources from fermentative media and the physicochemical parameters for growth were established by Plackett-Burman analysis, and the main parameters that affect extracellular inulinase yield were identified. In this study, the quantitative effect of the carbon to nitrogen ratio in the fermentative medium and the growth temperature were studied and optimized using central composite design and response surface methodology. METHODS AND RESULTS: On the basis of optimization, the maximum extracellular inulinase activity was achieved when 2·5-6·5% buckwheat flour was used as a single carbon source and 4·6-5·0% yeast extract was used as nitrogen source, by submerged cultivation, after 48 h at an incubation temperature between 15 and 27·5°C. CONCLUSIONS: Under the fermentative conditions established in this study, a maximum extracellular inulinase yield of 1·8 UI ml⁻¹ was achieved. Rhizoctonia ssp. strain can be used for extracellular inulinase production. Also, buckwheat flour proved to be an inexpensive and abundant substrate suitable for obtaining inulinase. SIGNIFICANCE AND IMPACT OF THE STUDY: Inulinases are versatile tools for biotechnology as they can be used for a wide range of applications, including production of bioethanol, fructose syrup and inulo-oligosaccharides, lactic acid, citric acid and butanediol.