Hyphal Cell Wall Research Articles

Microwave-Assisted Green Synthesis and Characterization of Silver Nanoparticles Using Melia azedarach for the Management of Fusarium Wilt in Tomato.

These days, research in agriculture is focusing on the theme of sustainability along with protection of agriculture produce. Nanotechnology in the agriculture sector aims for the enhancement of agricultural produce and the reduction of pesticides through providing innovative agrochemical agents and their novel delivery mechanisms. The current investigation involved the green synthesis of silver nanoparticles (AgNPs) from the aqueous leaf extract of Melia azedarach by following a microwave-assisted method to control Fusarium oxysporum, the causal agent of tomato wilt. Biosynthesized Melia leaf extract (MLE)-AgNPs were characterized by UV-visible spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), energy dispersive X-ray (EDX) spectrometry, dynamic light scattering (DLS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and zeta potential analysis. The intensity of the peak at 434 nm in UV-vis spectra, attributed to the surface plasmon resonance of MLE-AgNPs, changes with reaction parameters. TEM exhibits spherical shaped nanoparticles with an average particle size range from 12 to 46 nm. Efficient inhibition of F. oxysporum, the causal agent of tomato wilt, was achieved after exposure to MLE-AgNPs both in vivo and in vitro. In vitro studies exhibited repressed fungal mycelial growth with 79–98% inhibition as compared to the control. Significant increases in growth parameters of tomato seedlings were observed after treatment with biosynthesized nanoparticles as compared to F. oxysporum-infected plants grown without them under greenhouse conditions. Furthermore, SEM imaging was done to reveal the prominent damage on the cell wall of hyphae and spores after MLE-AgNP treatment. Propidium iodide (PI) staining of mycelium indicated the extent of cell death, causing irretrievable damage and disintegration of cellular membranes by altering the membrane permeability. Also, 2′,7′-dichlorofluorescin diacetate (DCFH-DA) fluorescence specifies intracellular reactive oxygen species (ROS) production in F. oxysporum after treatment with MLE-AgNPs. The current investigation suggested that biosynthesized nanoparticles can revolutionize the field of plant pathology by introducing an environment-friendly approach for disease management and playing a potential part in agriculture industry. However, to date, little work has been done to integrate nanotechnology into phytopathology so, this area of research is in need of adoption and exploration for the management of plant diseases.

Pathways That Synthesize Phosphatidylethanolamine Impact Candida albicans Hyphal Length and Cell Wall Composition through Transcriptional and Posttranscriptional Mechanisms.

Candida albicans is a leading cause of systemic bloodstream infections, and synthesis of the phospholipid phosphatidylethanolamine (PE) is required for virulence. The psd1Δ/Δ psd2Δ/Δ mutant, which cannot synthesize PE by the cytidine diphosphate diacylglycerol (CDP-DAG) pathway, is avirulent in the mouse model of systemic candidiasis. Similarly, an ept1Δ/Δ mutant, which cannot produce PE by the Kennedy pathway, exhibits decreased kidney fungal burden in systemically infected mice. Conversely, overexpression of EPT1 results in a hypervirulent phenotype in this model. Thus, mutations that increase PE synthesis increase virulence, and mutations that decrease PE synthesis decrease virulence. However, the mechanism by which virulence is regulated by PE synthesis is only partially understood. RNA sequencing was performed on strains with deficient or excessive PE biosynthesis to elucidate the mechanism. Decreased PE synthesis from loss of EPT1 or PSD1 and PSD2 leads to downregulation of genes that impact mitochondrial function. Losses of PSD1 and PSD2, but not EPT1, cause significant increases in transcription of glycosylation genes, which may reflect the substantial cell wall defects in the psd1Δ/Δ psd2Δ/Δ mutant. These accumulated defects could contribute to the decreased virulence observed for mutants with deficient PE synthesis. In contrast to mutants with decreased PE synthesis, there were no transcriptional differences between the EPT1 overexpression strain and the wild type, indicating that the hypervirulent phenotype is a consequence of posttranscriptional changes. It was found that overexpression of EPT1 causes increased chitin content and increased hyphal length. These phenotypes may help to explain the previously observed hypervirulence in the EPT1 overexpressor.

Analysis of the Bioactive Metabolites of the Endangered Mexican Lost Fungi Campanophyllum – A Report from India

Meghalaya, (in India), in the region of the mega-biodiversity hotspots, is home to a plethora of wild mushrooms. The present study concerns the exploration of the order Agaricales, which includes rare gilled mushrooms considered endangered under IUCN A4c criteria, due to the declining habitat. Electron microscopy of the gill sections revealed an abundance of clamp connections, hyphal cell walls, cystidia, and basidia. This rare species which belongs to the family Cyphellaceae, exhibits morphological and molecular differences from the Cyphella spp. Phylogenetic analysis revealed that it formed a clade under the genus Campanophyllum of the order Agaricales, confirmed by both Neighbor Joining (NJ) and Bayesian phylogenetic analysis. Being nutritionally potent along with its efficient antioxidant value, the fungal extract shows significant rise of two-fold in the antimicrobial activity along with the commercial antibiotics. The compound, Phenol, 2, 4-bis (1, 1-Dimethylethyl) (2, 4-DTBP) showed in ample range in the fungal extract along with aliphatic hydrocarbons, terpene, alcohol and volatile organic compounds on further characterization in GCMS. The present study indicates the endangered Campanophyllum proboscideum could be a rich source of natural antioxidants and an effective pharmaceutical agent.

Production of melanin pigments in saprophytic fungi in vitro and during infection.

Melanins are one of the great natural pigments produced by a wide variety of fungal species that promote fitness and cell survival in diverse hostile environments, including during mammalian infection. In this study, we sought to demonstrate the production of melanin in the conidia and hyphae of saprophytic fungi, including dematiaceous and hyaline fungi. We showed that a melanin-specific monoclonal antibody (MAb) avidly labeled the cell walls of hyphae and conidia, consistent with the presence of melanin in these structures, in 14 diverse fungal species. The conidia of saprophytic fungi were treated with proteolytic enzymes, denaturant, and concentrated hot acid to yield dark particles, which were shown to be stable free radicals, consistent with their identification as melanins. Samples obtained from patients with fungal keratitis due to Fusarium falciforme, Aspergillus fumigatus, Aspergillus flavus, Curvularia lunata, Exserohilum rostratum, or Fonsecaea pedrosoi were found to be intensely labeled by the melanin-specific MAb at the fungal hyphal cell walls. These results support the hypothesis that melanin is a common component that promotes survival under harsh conditions and facilitates fungal virulence. Increased understanding of the processes of melanization and the development of methods to interfere with pigment formation may lead to novel approaches to combat these complex pathogens that are associated with high rates of morbidity and mortality.

Candida albicans cell wall integrity transcription factors regulate polymicrobial biofilm formation with Streptococcus gordonii.

Polymicrobial biofilms play important roles in oral and systemic infections. The oral plaque bacterium Streptococcus gordonii is known to attach to the hyphal cell wall of the fungus Candida albicans to form corn-cob like structures in biofilms. However, the role of C. albicans in formation of polymicrobial biofilms is not completely understood. The objective of this study was to determine the role of C. albicans transcription factors in regulation of polymicrobial biofilms and antibiotic tolerance of S. gordonii. The proteins secreted by C. albicans and S. gordonii in mixed planktonic cultures were determined using mass spectrometry. Antibiotic tolerance of S. gordonii to ampicillin and erythromycin was determined in mixed cultures and mixed biofilms with C. albicans. Additionally, biofilm formation of S. gordonii with C. albicans knock-out mutants of 45 transcription factors that affect cell wall integrity, filamentous growth and biofilm formation was determined. Furthermore, these mutants were also screened for antibiotic tolerance in mixed biofilms with S. gordonii. Analysis of secreted proteomes resulted in the identification of proteins being secreted exclusively in mixed cultures. Antibiotic testing showed that S. gordonii had significantly increased survival in mixed planktonic cultures with antibiotics as compared to single cultures. C. albicans mutants of transcription factors Sfl2, Brg1, Leu3, Cas5, Cta4, Tec1, Tup1, Rim101 and Efg1 were significantly affected in mixed biofilm formation. Also mixed biofilms of S. gordonii with mutants of C. albicans transcription factors, Tec1 and Sfl2, had significantly reduced antibiotic tolerance as compared to control cultures. Our data indicates that C. albicans may have an important role in mixed biofilm formation as well as antibiotic tolerance of S. gordonii in polymicrobial biofilms. C. albicans may play a facilitating role than being just an innocent bystander in oral biofilms and infections.

Role of glomalin in soil carbon storage and its variation across land uses in temperate Himalayan regime

Abstract Root symbiont arbuscular mycorrhizal fungi (AMF) are ubiquitous in soils of terrestrial ecosystems and play a major role in determining the soil structure dynamics. Glomalin related soil protein (GRSP) produced in the hyphal cell walls is a store house of carbon that influences aggregate formation, stabilization and contributes in soil carbon sequestration. In this work, the variation in the glomalin concentrations was studied across soil of temperate Himalayan regime, its potential role in carbon storage and soil aggregation under different cropping systems. Aggregate stability showed a positive correlation with soil organic carbon (r = 0.966; p

Life at the edge – the cytology and physiology of the biotroph to necrotroph transition in Hymenoscyphus fraxineus during lesion formation in ash

The progress of colonization of ash stems from ascospore inocula of Hymenoscyphus fraxineus was examined by light and electron microscopy. The main aim of the study was to characterize the cytology of the biotroph to necrotroph transition during lesion formation. Following direct penetration into epidermal cells, the fungus produced intracellular hyphae that invaded up to five cells before plant cells died. A lack of close attachment between the hyphal cell wall and plant cell membrane was revealed by plasmolysis of epidermal cells. Plant cells died at the centre of the infection but hyphae at the edge were typically found in living plant cells even around large lesions. During biotrophic invasion, the cytoplasm of penetrated plant cells showed very little response despite the plant cell membrane being in direct contact with the fungal cell wall. Before plant cell death, dark staining of the cytoplasm and proliferation of small vesicles was noted, but organelles retained normal ultrastructure. Dead plant cells contained dark brown, osmiophilic droplets. Penetration between epidermal or collenchyma cells was usually targeted to shared pits and involved constriction of hyphae. The transition to necrotrophy was not associated with a clear change in hyphal morphology. Biotrophic intracellular hyphae contained dense cytoplasm but hyphae in dead plant cells were more vacuolated. Remarkably little plant cell wall degradation was observed despite the fungus penetrating up to 18 cells deep into stem tissue. Features of the development of the ash dieback fungus are compared with other hemibiotrophic pathogens.

Inhibition of Candida albicans morphogenesis by chitinase from Lactobacillus rhamnosus GG

Lactobacilli have been evaluated as probiotics against Candida infections in several clinical trials, but with variable results. Predicting and understanding the clinical efficacy of Lactobacillus strains is hampered by an overall lack of insights into their modes of action. In this study, we aimed to unravel molecular mechanisms underlying the inhibitory effects of lactobacilli on hyphal morphogenesis, which is a crucial step in C. albicans virulence. Based on a screening of different Lactobacillus strains, we found that the closely related taxa L. rhamnosus, L. casei and L. paracasei showed stronger activity against Candida hyphae formation compared to other Lactobacillus species tested. By exploring the activity of purified compounds and mutants of the model strain L. rhamnosus GG, the major peptidoglycan hydrolase Msp1, conserved in the three closely related taxa, was identified as a key effector molecule. We could show that this activity of Msp1 was due to its ability to break down chitin, the main polymer in the hyphal cell wall of C. albicans. This identification of a Lactobacillus-specific protein with chitinase activity having anti-hyphal activity will assist in better strain selection and improved application in future clinical trials for Lactobacillus-based Candida-management strategies.

Copper tolerance of the soft-rot fungus Phialophora malorum grown in-vitro revealed by microscopy and global protein expression

In this study, we used proteomics in conjunction with microscopy to study differences in the proteome and hyphal morphology of the copper tolerant soft rot fungus Phialophora malorum grown in media containing 0.064, 0.64% Cu as CuSO4. Unique proteins were found in the control and the copper-treated (0.064% CuSO4) samples. Of five unique proteins found in the 0.064% CuSO4 treated cultures, ATP synthase subunit alpha is considered to play an important role in copper tolerance as it is involved in the biosynthesis of fatty acids and steroids and may relate to morphological changes associated with hyphal cell walls of the fungus when grown in the presence of copper. ICP-AES analyses showed total mycelial Cu to increase with media Cu with 5246- and 16535 μg Cu/g dry wt mycelia respectively found in 0.064 and 0.64% Cu-cultures after 6 weeks growth. Rubeanic acid staining of 0.064% mycelia showed Cu bound in intracellular bodies while most Cu was found as extracellular precipitates on the surfaces of hyphae in 0.64% Cu. SEM showed hyphal surfaces enrobed in fibrillar polysaccharides to which Cu was bound.

Benzothiazole inhibits the growth of Phytophthora capsici through inducing apoptosis and suppressing stress responses and metabolic detoxification

Benzothiazole (BZO) is an antimicrobial secondary metabolite volatilized by many plants and microbes. However, the mechanism of BZO against phytopathogens is still unclear. Here, we found that BZO has antimicrobial activity against the oomycete pathogen Phytophthora capsici. Transcriptome and proteome analyses demonstrated that BZO significantly suppressed the expression of genes and proteins involved in morphology, abiotic stress defense and detoxification, but induced the activity of apoptosis. Annexin V-FITC/PI staining confirmed that the process of apoptosis was significantly induced by BZO at concentration of 150 mg L−1. FITC-phalloidin actin-cytoskeleton staining combined with hyphal cell wall staining and hyphal ultrastructure studies further confirmed that BZO disrupted the cell membrane and hyphal morphology through disrupting the cytoskeleton, eventually inhibiting the growth of hyphae. These data demonstrated that BZO has multiple modes of action and may act as potential leading compound for the development of new oomycete fungicides. These results also showed that the combination of transcriptomic and proteomic approaches was a useful method for exploring the novel antifungal mechanisms of natural compounds.

Cell wall structure of secreted laccase-silenced strain in Lentinula edodes

Laccase1 (Lcc1) is abundantly secreted from vegetative mycelia into culture medium by Lentinula edodes. Down-regulation of lcc1 in L. edodes results in abnormal hyphal structure and thinner cell wall in mycelia. In this study, we observed the effects of Lcc1 on the hyphal morphology and cell wall structure of L. edodes. A thick cell wall and fibrous layer were clearly observed in the lcc1-silenced strain ivrL1#32, when purified Lcc1 (0.1 mU/mL) was added to the culture medium. The ratio of cell wall polysaccharide contents was compared between the ivrL1#32 strain and the wild-type (WT) strain SR-1, revealing that levels of the alkali soluble β-1,3-1,6-glucan were significantly lower in the lcc1-silenced strain than in the WT strain. Chronological analysis revealed that chitin content in the cell wall did not increase over time, but that the alkali soluble β-1,3-1,6-glucan content increased after Lcc1 secretion in the WT. Taken together, these data suggest that the increased level of β-1,3-1,6-glucan induced by Lcc1 in the mycelial cell wall contributes to increased cell wall thickness and strength.

Novel Formulated Zinc Oxide Nanoparticles Reduce Hwp1 Gene Expression Involved in Biofilm Formation in Candida albicans with Minimum Cytotoxicity Effect on Human Cells

Background: Drug resistance in Candida species, has emerged as a major problem in the public health system worldwide. Application of nanoparticles is proposed as a novel and potential agent for reduction of drug resistance burdens. Objectives: The current study was conducted to evaluate the effects of zinc oxide nanoparticles (ZnO NPs containing chitosan and linoleic acid) on hyphae cell wall proteins (Hwp1) gene expression, a crucial gene in pathogenicity of Candida albicans, and cytotoxicity on human hepatocyte carcinoma (HepG2) cells as well as the production of Reactive Oxygen Species (ROS). Methods: The effects of novel ZnO NPs on expression of Hwp1 gene of C. albicans was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR) in comparison to fluconazole as a standard drug. Reactive Oxygen species production was examined in macrophages treated with ZnO NPs relative to non-treated cells. Also, the cytotoxicity effects of ZnO NPs were assessed using the MTT assay against HepG2 cell line. Results: The findings indicated that ZnO NPs significantly decreased the level of Hwp1 gene expression in standard and clinical isolates of C. albicans. Increased level of ROS production in macrophages was found in the presence of ZnO NPs in concentration-dependent manner compared to the control group without exposure of ZnO NPs (P = 0.001). Furthermore, ZnO NPs did not show cytotoxicity activity on HepG2 cells at different concentrations (P > 0.05). Conclusions: Taken together, the newly synthesized ZnO NPs may be a suitable candidate for inhibition of the critical gene responsible for biofilm dispersion and the control of Candida infection with limited cytotoxicity on human cells. However, more studies are required for support of its effect in vitro and in vivo.

The Gpr1-regulated Sur7 family protein Sfp2 is required for hyphal growth and cell wall stability in the mycoparasite Trichoderma atroviride

Mycoparasites, e.g. fungi feeding on other fungi, are prominent within the genus Trichoderma and represent a promising alternative to chemical fungicides for plant disease control. We previously showed that the seven-transmembrane receptor Gpr1 regulates mycelial growth and asexual development and governs mycoparasitism-related processes in Trichoderma atroviride. We now describe the identification of genes being targeted by Gpr1 under mycoparasitic conditions. The identified gene set includes a candidate, sfp2, encoding a protein of the fungal-specific Sur7 superfamily, whose upregulation in T. atroviride upon interaction with a fungal prey is dependent on Gpr1. Sur7 family proteins are typical residents of membrane microdomains such as the membrane compartment of Can1 (MCC)/eisosome in yeast. We found that GFP-labeled Gpr1 and Sfp2 proteins show partly overlapping localization patterns in T. atroviride hyphae, which may point to shared functions and potential interaction during signal perception and endocytosis. Deletion of sfp2 caused heavily altered colony morphology, defects in polarized growth, cell wall integrity and endocytosis, and significantly reduced mycoparasitic activity, whereas sfp2 overexpression enhanced full overgrowth and killing of the prey. Transcriptional activation of a chitinase specific for hyphal growth and network formation and strong downregulation of chitin synthase-encoding genes were observed in Δsfp2. Taken together, these findings imply crucial functions of Sfp2 in hyphal morphogenesis of T. atroviride and its interaction with prey fungi.

Protein Deiminase 4 and CR3 Regulate Aspergillus fumigatus and β-Glucan-Induced Neutrophil Extracellular Trap Formation, but Hyphal Killing Is Dependent Only on CR3.

Neutrophil extracellular trap (NET) formation requires chromatin decondensation before nuclear swelling and eventual extracellular release of DNA, which occurs together with nuclear and cytoplasmic antimicrobial proteins. A key mediator of chromatin decondensation is protein deiminase 4 (PAD4), which catalyzes histone citrullination. In the current study, we examined the role of PAD4 and NETosis following activation of neutrophils by A. fumigatus hyphal extract or cell wall β-glucan (curdlan) and found that both induced NET release by human and murine neutrophils. Also, using blocking antibodies to CR3 and Dectin-1 together with CR3-deficient CD18−/− and Dectin-1−/− murine neutrophils, we found that the β-glucan receptor CR3, but not Dectin-1, was required for NET formation. NETosis was also dependent on NADPH oxidase production of reactive oxygen species (ROS). Using an antibody to citrullinated histone 3 (H3Cit) as an indicator of PAD4 activity, we show that β-glucan stimulated NETosis occurs in neutrophils from C57BL/6, but not PAD4−/− mice. Similarly, a small molecule PAD4 inhibitor (GSK484) blocked NET formation by human neutrophils. Despite these observations, the ability of PAD4−/− neutrophils to release calprotectin and kill A. fumigatus hyphae was not significantly different from C57BL/6 neutrophils, whereas CD18−/− neutrophils exhibited an impaired ability to perform both functions. We also detected H3Cit in A. fumigatus infected C57BL/6, but not PAD4−/− corneas; however, we found no difference between C57BL/6 and PAD4−/− mice in either corneal disease or hyphal killing. Taken together, these findings lead us to conclude that although PAD4 together with CR3-mediated ROS production is required for NET formation in response to A. fumigatus, PAD4-dependent NETosis is not required for A. fumigatus killing either in vitro or during infection.

Ganoderma lucidum phosphoglucomutase is required for hyphal growth, polysaccharide production, and cell wall integrity.

Phosphoglucomutase (pgm) is an important enzyme in carbohydrate metabolism that is located at the branching point between glycolysis and the Leloir pathway. pgm catalyzes the reversible conversion reaction between glucose-6-phosphate (Glc-6-P) and glucose-1-phosphate (Glc-1-P). The glpgm gene was cloned in Escherichia coli, and the recombinant pgm protein from Ganoderma lucidum was purified in this study. The activity of native pgm was also detected to demonstrate that this predicted gene was functional in G. lucidum. Interestingly, silencing the glpgm gene in the fungus reduced hyphal growth. Moreover, glpgm silencing was associated with declining extracellular polysaccharide (EPS) production (approximately 20-40% of that in the WT strain) and increasing intracellular polysaccharide (IPS) production (approximately 1.7-fold that in the WT strain). Additionally, in our research, cell wall components were also shown to differ according to the glpgmi strain. Compared with WT, chitin significantly increased by 1.5-fold; however, the content of β-1,3-glucan was observably reduced to 60-70% that of the WT. Further research showed that the cell wall component changes were associated with the transcription of related genes. These findings provide references for further study on the potential physiological function of pgm in G. lucidum.

Vital role for cyclophilin B (CypB) in asexual development, dimorphic transition and virulence of Beauveria bassiana

Cyclophilin B (CypB) was previously revealed as one of many putative secretory proteins in the transcriptome of Beauveria bassiana infection to a lepidopteran pest. Here we show a main localization of CypB in hyphal cell walls and septa and its essential role in the in vitro and in vivo asexual cycles of the fungal insect pathogen. Deletion of cypB reduced colony growth by 16–42% on two rich media and 30 scant media with different carbon or nitrogen sources. The deletion mutant suffered a delayed conidiation on a standard medium and a final 47% reduction in conidial yield, accompanied with drastic transcript depression of several key genes required for conidiation and conidial maturation. The mutant conidia required 10h longer to germinate 50% at optimal 25°C than wild-type conidia. Intriguingly, cultivation of the mutant conidia in a trehalose-peptone broth mimic to insect hemolymph resulted in 83% reduction in blastospore yield but only slight decrease in biomass level, indicating severe defects in transition of hyphae to blastospores. LT50 for the deletion mutant against Galleria mellonella larvae through normal cuticle infection was prolonged to 7.4d from a wild-type estimate of 4.7d. During colony growth, additionally, the deletion mutant displayed hypersensitivity to Congo red, menadione, H2O2 and heat shock but increased tolerance to cyclosporine A and rapamycin. All of changes were restored by targeted gene complementation. Altogether, CypB takes part in sustaining normal growth, aerial conidiation, conidial germination, dimorphic transition, stress tolerance and pathogenicity in B. bassiana.

Lipopeptides produced by Bacillus subtilis as new biocontrol products against fusariosis in ornamental plants.

In this study, we have investigated the effects of three lipopeptides (fengycin, surfactin and mycosubtilin) produced by different strains of Bacillus subtilis against the phytopathogenic fungi Fusarium oxysporum f. sp. iridacearum, which affects the ornamental bulb plant populations of Iris sp. The antifungal effects were tested using minimum inhibitory concentration assay, determination of mycelium growth and spore germination inhibition rates. Also, in vivo tests on infected rhizomes and scanning electron microscopy were employed. Mycosubtilin alone and in combination with fengycin or/and surfactin showed potent inhibitory activity at concentrations as low as 5μgml-1 which is 100 times lower compared to Topsin M, a common chemical fungicide frequently used against fusariosis in ornamental plants. An enhancement of mycosubtilin antifungal activity was observed when it was used in combination with surfactin due to a synergistic effect. At a concentration of 20μgml-1, mycosubtilin inhibited the growth of the mycelium up to 49% and the spore germination ability up to 26% in comparison to control. In addition, significant changes on the macro- and micro-morphology have been observed. The antifungal activity is related to the inhibition of spore germination and the irreversible damage of the hyphae cell wall. To the best of our knowledge, this is the first attempt to propose the lipopeptides as biopesticides against the fusariosis of ornamental plants.

Synthesis and Fungicidal Activity of (E)‐5‐[1‐(2‐Oxo‐1‐oxaspiro[4,5]dec/non‐3‐en‐3‐yl)ethylidene]‐2‐aminoimidazolin‐4‐one Derivatives

AbstractThe novel fungicidal agents, (E)‐5‐[1‐(2‐oxo‐1‐oxaspiro[4,5]dec/non‐3‐en‐3‐yl)ethylidene]‐2‐aminoimidazolin‐ 4‐one derivatives, were designed and synthesized in moderate to excellent yields in four steps using α‐hydroxyketone and diketene as raw materials and characterized by HR‐ESI‐MS, 1H NMR and X‐ray diffraction. The preliminary bioassay showed that some of these compounds, such as 5e, 6a, 6e, and 7 h exhibit 87.8%, 91.3%, 89.9% and 87.8% inhibition rates against Sclerotinia scleotiorum, 3b, 3c, 4c and 7 h exhibit 96.4%, 92.5%, 90.3% and 76.9% inhibition rates against Phytophthora capsici at the concentration of 50 µg/mL, respectively. These compounds exhibited significant fungicidal activities against S. scleotiorum and P. capsici with EC50 values of 2.56–11.60 µg/mL, and compounds 6e and 7 h exhibited weak inhibition against the spore germination of S. scleotiorum, while the spore germination of P. capsici was strongly inhibited by compound 7 h solution. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observation indicated that compound 7 h had a significant impact on the structure and function of the hyphal cell wall of P. capsici mycelium.

Alteration in the ultrastructural morphology of mycelial hyphae and the dynamics of transcriptional activity of lytic enzyme genes during basidiomycete morphogenesis.

The morphogenesis of macromycetes is a complex multilevel process resulting in a set of molecular-genetic, physiological-biochemical, and morphological-ultrastructural changes in the cells. When the xylotrophic basidiomycetes Lentinus edodes, Grifola frondosa, and Ganoderma lucidum were grown on wood waste as the substrate, the ultrastructural morphology of the mycelial hyphal cell walls differed considerably between mycelium and morphostructures. As the macromycetes passed from vegetative to generative development, the expression of the tyr1, tyr2, chi1, chi2, exg1, exg2, and exg3 genes was activated. These genes encode enzymes such as tyrosinase, chitinase, and glucanase, which play essential roles in cell wall growth and morphogenesis.

Synergic effect of combination of glycyrol and fluconazole against experimental cutaneous candidiasis due to Candida albicans.

In this study, we investigated the anti-fungal activity of glycyrol, a coumarine isolated from licorice (Glycyrrhizae Radix), in a murine model of cutaneous candidiasis caused by Candida albicans. Compared to the infected sites, located on the mice's back, of the untreated control mice, the infected sites treated with glycyrol had reduced CFU (colony forming unit) values up to 60 and 85.5% at 20 and 40μg/mouse of glycyrol, respectively (P<0.01). The antifungal activity of glycyrol was synergistically increased when glycyrol (10μg/mouse) was combined with fluconazole (10μg/mouse), demonstrating that the combination therapy is approximately 4 times more effective than fluconazole alone at 20μg/mouse (P<0.01). Additionally, the combination activity was 1.65 times greater than the antifungal activity of fluconazole alone at 40μg/mouse (P<0.05). In seeking glycyrol's antifungal mechanism, we determined that glycyrol inhibited hyphal induction and cell wall adherence of C. albicans. Thus, it is very likely that, by damaging the cell wall, glycyrol helps fluconazole invade C. albicans more readily and attack fluconazole's target in the fungus membrane. In summary, our data indicate that glycyrol may contribute to the development of a novel agent that possesses antifungal activity against cutaneous candidiasis.