Mycelial Extracts Research Articles

Venturicidin A Is a Potential Fungicide for Controlling Fusarium Head Blight by Affecting Deoxynivalenol Biosynthesis, Toxisome Formation, and Mitochondrial Structure.

Fusarium graminearum, which causes Fusarium head blight (FHB) in cereals, is one of the most devastating fungal diseases by causing great yield losses and mycotoxin contamination. A major bioactive ingredient, venturicidin A (VentA), was isolated from Streptomyces pratensis S10 mycelial extract with an activity-guided approach. No report is available on antifungal activity of VentA against F. graminearum and effects on deoxynivalenol (DON) biosynthesis. Here, VentA showed a high antagonistic activity toward F. graminearum with an EC50 value of 3.69 μg/mL. As observed by scanning electron microscopy, after exposure to VentA, F. graminearum conidia and mycelia appeared abnormal. Different dyes staining revealed that VentA increased cell membrane permeability. In growth chamber and field trials, VentA effectively reduced disease severity of FHB. Moreover, VentA inhibited DON biosynthesis by reducing pyruvic acid, acetyl-CoA production, and accumulation of reactive oxygen species (ROS) and then inhibiting trichothecene (TRI) genes expression and toxisome formation. These results suggest that VentA is a potential fungicide for controlling FHB.

Nutritional value, elemental bioaccumulation and antioxidant activity of fruiting bodies and mycelial cultures of an unrecorded wild Lactarius hatsudake from Nanyue mountainous region in China

An unrecorded wild mushroom Lactarius hatsudake from Nanyue mountainous region in China was identified. Subsequently, comparative investigation on the nutritional value, elemental bioaccumulation, and antioxidant activity was performed in the fruiting body (FB) and mycelium (MY) samples of this species. It revealed that the contents of moisture (87.66 ± 0.16 g/100 g fw) and ash (6.97 ± 0.16 g/100 g dw) were significantly higher in FB, and the total carbohydrate, fat, and protein concentrations of FB were similar to those in MY. Among nutritionally important elements, FB possessed higher concentrations of potassium (37808.61 ± 1237.38 mg/kg dw), iron (470.69 ± 85.54 mg/kg dw), and zinc (136.13 ± 5.16 mg/kg dw), whereas MY was a better source of magnesium (1481.76 ± 18.03 mg/kg dw), calcium (2203.87 ± 69.61 mg/kg dw), and sodium (277.44 ± 22.93 mg/kg dw). According to the health risk estimation, FB might pose an aluminum-related health problem when a prolonged period of exposure, while MY was risk-free for consumers. The results of antioxidant capacity (1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assays) in FB and MY were within the range of 104.19 ± 5.70 mg ascorbic acid equivalents (AAE)/g to 169.50 ± 4.94 mg AAE/g, and half maximal effective concentration EC50 values ranged from 0.23 ± 0.01 mg/mL to 0.62 ± 0.05 mg/mL. The aqueous extracts of MY demonstrated a strong ABTS radical scavenging capacity with the highest AAE value.

Fibronectin-binding molecules of Scedosporium apiospermum: focus on adhesive events.

Scedosporium apiospermum is a widespread, emerging, and multidrug-resistant filamentous fungus that can cause localized and disseminated infections. The initial step in the infection process involves the adhesion of the fungus to host cells and/or extracellular matrix components. However, the mechanisms of adhesion involving surface molecules in S. apiospermum are not well understood. Previous studies have suggested that the binding of fungal receptors to fibronectin enhances its ability to attach to and infect host cells. The present study investigated the effects of fibronectin on adhesion events of S. apiospermum. The results revealed that conidial cells were able to bind to both immobilized and soluble human fibronectin in a typically dose-dependent manner. Moreover, fibronectin binding was virtually abolished in trypsin-treated conidia, suggesting the proteinaceous nature of the binding site. Western blotting assay, using fibronectin and anti-fibronectin antibody, evidenced 7 polypeptides with molecular masses ranging from 55 to 17kDa in both conidial and mycelial extracts. Fibronectin-binding molecules were localized by immunofluorescence and immunocytochemistry microscopies at the cell wall and in intracellular compartments of S. apiospermum cells. Furthermore, a possible function for the fibronectin-like molecules of S. apiospermum in the interaction with host lung cells was assessed. Conidia pre-treated with soluble fibronectin showed a significant reduction in adhesion to either epithelial or fibroblast lung cells in a classically dose-dependent manner. Similarly, the pre-treatment of the lung cells with anti-fibronectin antibodies considerably diminished the adhesion. Collectively, the results demonstrated the presence of fibronectin-binding molecules in S. apiospermum cells and their role in adhesive events.

Enhanced podophyllotoxin production of endophyte Fusarium proliferatum TQN5T by host extract and phenylalanine.

Fermentation technology using endophytes is considered a potential alternative approach for producing pharmaceutical compounds like podophyllotoxin (PTOX). In this study, fungus TQN5T (VCCM 44284) was selected from endophytic fungi isolated from Dysosma versipellis in Vietnam for PTOX production through TLC. The presence of PTOX in TQN5T was further confirmed by HPLC. Molecular identification indicated TQN5T as Fusarium proliferatum with 99.43% identity. This result was asserted by morphological characteristics such as white cottony, filamentous colony, layer and branched mycelium, and clear hyphae septa. Cytotoxic assay indicated both biomass extract and culture filtrate of TQN5T presented strong cytotoxicity on LU-1 and HepG2 with IC50 of 0.11, 0.20, 0.041, and 0071, respectively, implying anti-cancer compounds were accumulated in the mycelium and secreted into the medium. Further, the production of PTOX in TQN5T was investigated in the fermentation condition supplemented with 10µg/ml of host plant extract or phenylalanine as elicitors. The results revealed a significantly higher amount of PTOX in the PDB + PE and PDB + PA at all studied time points in comparison with PDB (control). Especially, after 168h of culture, PTOX content in the PDB with plant extract reached the peak with 314µg/g DW which is 10% higher than the best yield of PTOX in previous studies, denoting F. proliferatum TQN5T as a promising PTOX producer. This is the first study on enhancing the PTOX production in endophytic fungi by supplementing phenylalanine-a precursor for PTOX biosynthesis in plants into fermented media, suggesting a common PTOX biosynthetic pathway between host plant and endophytes. KEY POINTS: • Fusarium proliferatum TQN5T was proven for PTOX production. • Both mycelia extract and spent broth extract of Fusarium proliferatum TQN5T presented strong cytotoxicity on cancer cell lines LU-1 and HepG2. • The supplementation of 10µg/ml host plant extract and phenylalanine into fermentation media of F. proliferatum TQN5T improved the yield of PTOX.

Preventative Effects of Cordyceps cicadae Mycelial Extracts on the Early-Stage Development of Cataracts in UVB-Induced Mice Cataract Model.

Cataracts, a prevalent age-related eye condition, pose a significant global health concern, with rising rates due to an aging population and increased digital device usage. In Taiwan, cataract prevalence is particularly high, reaching up to 90% among individuals aged 70 and above. The lens of the eye absorbs short-wave light, which can lead to oxidative stress in lens epithelial cells and contribute to cataract formation. Exposure to ultraviolet (UV) light further exacerbates the risk of cataracts by generating reactive oxygen species. Heat-shock proteins (HSPs), involved in protein maintenance and repair, have been linked to cataract development. Cordyceps cicadae (C. cicadae), a traditional Chinese medicine, has a long history of use and is known for its pharmacological effects. N6-(2-hydroxyethyl) adenosine (HEA), a bioactive compound found in C. cicadae, exhibits anti-inflammatory, immunomodulatory, and neuroprotective properties. Previous studies have shown that C. cicadae mycelial extracts improve dry eye disease and reduce intraocular pressure in animal models. Additionally, C. cicadae possesses antioxidant properties, which are beneficial for combating cataract formation. In this study, we aim to evaluate the preventive efficacy of C. cicadae mycelial extracts in UV-induced cataract development. By investigating the ameliorative effects of C. cicadae on eye diseases and its potential role in ocular health improvement, we hope to uncover new options for cataract prevention and provide insights into the mechanisms of action. The findings of this research could provide a novel approach for nutritional supplements targeting cataract prevention, offering potential benefits in the field of ocular health.

Chemical analysis and antitubercular activity evaluation of the dried mycelial powders of the basidiomycete Ganoderma australe TBRC-BCC 22314

The isolation of lanostane triterpenoids possessing significant anti-tuberculosis (anti-TB) activity from mycelial cultures of the basidiomycete Ganoderma australe strain TBRC-BCC 22314 was previously reported. To demonstrate the potential of the dried mycelial powder for utilization in anti-TB medicinal products, its authentic chemical analysis was performed. Considering the possibility of the changes in the lanostane compositions and anti-TB activity by sterilization, both autoclave treated and non-autoclaved mycelial powder materials were chemically investigated. The study led to the identification of the lanostanes responsible for the activity of the mycelial extract against Mycobacterium tuberculosis H37Ra. The anti-TB activity of the extracts from autoclaved and non-autoclaved mycelial powders were the same (MIC 3.13 μg/mL). However, the analytical results revealed several unique chemical conversions of the lanostanes under the sterilization conditions. The most potent major lanostane, ganodermic acid S (1), was shown to be significantly active also against the extensively drug-resistant (XDR) strains of M. tuberculosis.

Psychrotolerant Strains of Phoma herbarum with Herbicidal Activity

The search for stress-tolerant producer strains is a key factor in the development of biological mycoherbicides. The aim of the study was to assess the herbicidal potential of phoma-like fungi. Morphological and physiological features of two Antarctic psychrotolerant strains 20-A7-1.M19 and 20-A7-1.M29 were studied. Multilocus sequence analysis was used to identify these strains. They happened to belong to Phoma herbarum Westend. The psychrotolerant properties of these strains were suggested not only by ecology, but also by their capability to grow in a wide temperature range from 5 °C to 35 °C, being resistant to high insolation, UV radiation, aridity, and other extreme conditions. It was shown that treatment with their cell-free cultural fugate, crude mycelium extract, and culture liquid significantly reduced the seed germination of troublesome weeds such as dandelion and goldenrod. Cell-free cultural fugate and culture liquid also led to the formation of chlorosis and necrotic spots on leaves. Thus, psychrotolerant strains P. herbarum 20-A7-1.M19 and 20-A7-1.M29 demonstrate high biotechnological potential. Our next step is to determine the structures of biologically active substances and to increase their biosynthesis, as well as the development of biological and biorational mycoherbicides. New mycoherbicides can reduce the chemical load on agroecosystems and increase the effectiveness of applied chemicals.

Ganoderma lucidum-Mediated Green Synthesis of Silver Nanoparticles with Antimicrobial Activity.

"Green chemistry" is a simple and easily reproductible method that provides nanoparticles characterized by better stability and good dispersion in an aqueous solution. Nanoparticles can be synthesized by algae, bacteria, fungi, and plant extracts. Ganoderma lucidum is a commonly used medicinal mushroom with distinctive biological properties, such as antibacterial, antifungal, antioxidant, anti-inflammatory, anticancer, etc. In this study, aqueous mycelial extracts of Ganoderma lucidum were used to reduce AgNO3 to form silver nanoparticles (AgNPs). The biosynthesized nanoparticles were characterized by UV-visible spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR) analysis. The maximum UV absorption was obtained at 420 nm, which represents the specific surface plasmon resonance band for biosynthesized silver nanoparticles. SEM images showed particles as predominantly spherical, while FTIR spectroscopic studies illustrated the presence of functional groups that can support the reducing of ion Ag+ to Ag(0). XRD peaks ratified the presence of AgNPs. The antimicrobial effectiveness of synthesized nanoparticles was tested against Gram-positive and Gram-negative bacterial and yeasts strains. The silver nanoparticles were effective against pathogens, inhibiting their proliferation, and thus reducing the risk to the environment and to public health.

Diversity of Mycogenic Oxide and Chalcogenide Nanoparticles: A Review.

Oxide and chalcogenide nanoparticles have great potential for use in biomedicine, engineering, agriculture, environmental protection, and other research fields. The myco-synthesis of nanoparticles with fungal cultures, their metabolites, culture liquids, and mycelial and fruit body extracts is simple, cheap and environmentally friendly. The characteristics of nanoparticles, including their size, shape, homogeneity, stability, physical properties and biological activity, can be tuned by changing the myco-synthesis conditions. This review summarizes the data on the diversity of oxide and chalcogenide nanoparticles produced by various fungal species under different experimental conditions.

An endophytic fungus Schizophyllum commune isolated from Panax ginseng enhances hairy roots growth and ginsenoside biosynthesis.

Using endophytic fungal elicitors to increase the accumulation of valuable secondary metabolites in plant tissue culture is an effective biotechnology strategy. In this study, a collection of 56 strains of endophytic fungi were isolated from different organs of cultivated Panax ginseng, of which seven strains can be symbiotically co-cultured with the hairy roots of P. ginseng. Further experiments observed that strain 3R-2, identified as endophytic fungus Schizophyllum commune, can not only infect hairy roots but also promote the accumulation of specific ginsenosides. This was further verified because S. commune colonization significantly affected the overall metabolic profile of ginseng hairy roots. By comparing the effects of S. commune mycelia and its mycelia extract (EM) on ginsenoside production in P. ginseng hairy roots, the EM was confirmed to be a relatively better stimulus elicitor. Additionally, the introduction of EM elicitor can significantly enhance the expressions of key enzyme genes of pgHMGR, pgSS, pgSE, and pgSD involved in the biosynthetic pathway of ginsenosides, which was deemed the most relevant factor for promoting ginsenosides production during the elicitation period. In conclusion, this study is the first to show that the EM of endophytic fungus S. commune can be considered as an effective endophytic fungal elicitor for increasing the biosynthesis of ginsenosides in hairy root cultures of P. ginseng.

Isolation of Fungi from Mangrove Ecosystem of Mumbai and Evaluation of their Antibacterial Potential

In the current study, fungi from the mangrove ecosystem of Mumbai were isolated and their metabolites were screened for antibacterial potential. Two weeks old broth and mycelium were extracted using chloroform and methanol. Antibacterial property of solvent extracts was evaluated at various concentrations (2 – 10 µg/ml) against Staphylococcus aureus, Bacillus subtilis, Enterococcus faecium, Enterococcus faecalis, Klebsiella pneumonia and Escherichia coli, by well diffusion method. Fungi isolated were identified as Aspergillus niger, Aspergillus flavus, Trichoderma harzianum, Cylindrocladium scoparium and Colletotrichum wuxiense. Results revealed that broth solvent extracts of isolates inhibited the growth of all gram-positive test bacteria, chloroform broth extract of Cylindrocladium scoparium, Colletotrichum wuxiense and ethanolic broth extract of Aspergillus flavus, Trichoderma harzianum exhibited antibacterial potential against gram negative test organisms. Chloroform and ethanol mycelium extracts of Trichoderma harzianum and Aspergillus flavus, respectively, exhibited 100% growth inhibition potential against all test organisms. The current investigation endorses the potent secondary metabolism of the identified isolates and their potential to synthesise antibacterial compounds.

Combination Treatment in Ulcerative Colitis using 5-Aminosalysilic Acid (5-ASA) and Polysaccharide Peptide of Indonesian Ganoderma lucidum Mycelium Extract

Background: Inflammatory bowel disease (IBD) is idiopathic disease characterized by chronic inflammation of the gastrointestinal tract. Polysaccharide peptide of Ganoderma lucidum mycelium extract (PPGL) is considerably a good option for adjunctive therapy of IBD. This study aims to evaluate the benefit of PPGL in patients treated with 5-ASA.Method: A retrospective observational cohort study was conducted to examine the medical records of 124 ulcerative colitis patients. There were 80 patients in intervention group who were treated with a combination of 5-ASA and PPGL, and there were 44 patients in the control group who were treated with 5-ASA only. Clinical and laboratory endpoints were observed at the baseline and after 30, 60, and 90 days. Clinical endpoints included abdominal pain, bloody diarrhea, aphthous stomatitis, and polyarthritis; meanwhile, laboratory endpoints included hemoglobin level, ESR, CRP, fecal calprotectin, M2-pyruvate kinase (MP2K), fecal culture, C. difficile culture, and colonoscopy results. Clinical trials conducted after patient enrollment with registry number ClinicalTrials.gov NCT04029649.Results: On day-30 there was a significant difference between intervention group 45.6% and control group 2.3% in abdominal pain complaints (p 0.001). Moreover, laboratory parameters of fecal calprotectin (p 0.001), fecal MP2K (p = 0.015), and hemoglobin (p 0.001) were considerably better in intervention group on day-30. These differences were consistently found on day 60 and 90.Conclusion: The study implies potential correlation between PPGL administration and improvement of clinical and laboratory endpoints up to 90 days. A larger randomized, blinded, prospective study is required to confirm these effects in ulcerative colitis.

Acetylcholine Esterase Inhibitory Effect, Antimicrobial, Antioxidant, Metabolomic Profiling, and an In Silico Study of Non-Polar Extract of The Halotolerant Marine Fungus Penicillium chrysogenum MZ945518.

Major health issues, such as the rise in oxidative stress, incidences of Alzheimer's disease, and infections caused by antibiotic-resistant microbes, have prompted researchers to look for new therapeutics. Microbial extracts are still a good source of novel compounds for biotechnological use. The objective of the current work was to investigate marine fungal bioactive compounds with potential antibacterial, antioxidant, and acetylcholinesterase inhibitory effects. Penicillium chrysogenum strain MZ945518 was isolated from the Mediterranean Sea in Egypt. The fungus was halotolerant with a salt tolerance index of 1.3. The mycelial extract showed antifungal properties against Fusarium solani with an inhibitory percentage of 77.5 ± 0.3, followed by Rhizoctonia solani and Fusarium oxysporum with percentages of 52 ± 0.0 and 40 ± 0.5, respectively. The extract also showed antibacterial activity against both Gram-negative and Gram-positive bacterial strains using the agar diffusion technique. The fungal extract was significantly more effective with Proteus mirabilis ATCC 29906 and Micrococcus luteus ATCC 9341; inhibition zones recorded 20 and 12 mm, respectively, compared with the antibiotic gentamycin, which recorded 12 and 10 mm, respectively. The antioxidant activity of the fungus extract revealed that it successfully scavenged DPPH free radicals and recorded an IC50 of 542.5 µg/mL. Additionally, it was capable of reducing Fe3+ to Fe2+ and exhibiting chelating ability in the metal ion-chelating test. The fungal extract was identified as a crucial inhibitor of acetylcholinesterase with an inhibition percentage of 63% and an IC50 value of 60.87 µg/mL. Using gas chromatography-mass spectrometry (GC/MS), 20 metabolites were detected. The most prevalent ones were (Z)-18-octadec-9-enolide and 1,2-Benzenedicarboxylic acid, with ratios of 36.28 and 26.73%, respectively. An in silico study using molecular docking demonstrated interactions between the major metabolites and the target proteins, including: DNA Gyrase, glutathione S-transferase, and Acetylcholinesterase, confirming the extract's antimicrobial and antioxidant activity. Penicillium chrysogenum MZ945518, a halotolerant strain, has promising bioactive compounds with antibacterial, antioxidant, and acetylcholinesterase inhibitory activities.

The mycelium of the Trametes versicolor synn. Coriolus versicolor (Turkey tail mushroom) exhibit anti-melanoma activity in vitro

Melanoma is one of the most aggressive forms of skin cancer and is characterized by high metastatic potential. Despite improvements in early diagnosis and treatment, the mortality rate among metastatic melanoma patients continues to represent a significant clinical challenge. Therefore, it is imperative that we search for new forms of treatment. Trametes versicolor is a mushroom commonly used in Chinese traditional medicine due to its numerous beneficial properties. In the present work, we demonstrate T. versicolor fruiting body and mycelium ethanol extracts exhibit potent cytotoxic activity towards A375 (IC50 = 663.3 and 114.5 µg/mL respectively) and SK-MEL-5 (IC50 = 358.4 and 88.6 µg/mL respectively) human melanoma cell lines. Further studies revealed that T. versicolor mycelium extract induced apoptotic cell death and poly (ADP-ribose) polymerase cleavage, upregulated the expression of autophagy-associated marker LC3-II, increased the presentation of major histocompatibility complex II and expression of programmed death-ligand receptor, and inhibited cell migration in SK-MEL-5 cells. Therefore, our present findings highlight the therapeutic potential of T. versicolor mycelium extract for the treatment of melanoma and merit further study.

Features of the Formation of Sensitive Films Based on Mycelium of Higher Fungi for Surface and Plate Acoustic Waves Gas Sensors.

A comparative analysis of the responses of two types of acoustic waves (surface SAW and plate APW) with close frequencies and the same type of waves (SAW) with different frequencies toward various liquid vapors (water, acetone, ethanol) was carried out in this paper. Two types of films based on mycelium of higher fungus Ganoderma lucidum (Curtis) P. Karst (G. lucidum) prepared by various methods were used as sensitive coatings. These films were based on G. lucidum mycelium ethanolic (48% v/v) homogenizate (MEGl) and extract (EGl). A film deposition procedure compatible with acoustic devices technology was developed. Various piezoelectric substrates (YX-LiNbO3, 128 YX-LiNbO3) were used for appropriate acoustic delay lines production. It was found that additional SAW and APW attenuation associated with the appearance of mycelium films on the surface of the acoustic waveguide is two times greater for MEGL than for EGL films in the frequency range of 20-80 MHz The changes in acoustic wave amplitude and phase due to vapor absorption were measured and compared with each other, taking into account the differences in geometry of the samples. It was found that the phase response of the SAW delay lines with EGL films is three times higher than one with the presence of MEGL films for water and ethanol vapors. The films used are demonstrated good reproducibility and long-term stability for at least 2 months. Based on the results obtained, it was concluded that MEGl film is not appropriate for use in high frequency SAW delay lines as a sensitive coating. However, both types of the films (MEGl and EGl) could be used as sensitive coatings for low frequency SAW and APW sensors based on corresponding delay lines. Additionally, it was found that the films used are not sensitive to acetone vapor. As a result of the work carried out, a technique for creating sensitive films based on the mycelium of higher fungi compatible with the planar technology of acoustoelectronic delay lines was developed. The possibility of using such films for the development of gas SAW and APW sensors was shown.

Promoting effects of <italic>Tulasnella</italic> sp. S7 metabolites on <italic>Dendrobium officinale </italic>seed germination

<p indent="0mm">The seeds of the orchid<italic> Dendrobium officinale</italic> symbiotically germinated with <italic>Tulasnella</italic> sp. S7. The S7 extracts promoted germination. In this study, the ethyl acetate fraction of the S7 mycelium extract (MyE-E) and the petroleum ether and ethyl acetate fractions of the S7 fermentation broth extract (FBE-P and FBE-E) were confirmed to be active by activity tracking. Thereafter, the FBE-E chemical constituents were isolated by column chromatography and preparative high-performance liquid chromatography, and their activities were detected. Twelve compounds were isolated and elucidated as diisobutyl phthalate (<bold>1</bold>), dibutyl phthalate (<bold>2</bold>), indole-3-carboxaldehyde (<bold>3</bold>), vanillic acid (<bold>4</bold>), isovanillic acid (<bold>5</bold>), syringic acid (<bold>6</bold>), <italic>trans</italic>-ferulic acid (<bold>7</bold>), 3,5-dihydroxybenzoic acid (<bold>8</bold>), 3,4-dihydroxybenzoic acid (<bold>9</bold>), indole-3-carboxylic acid (<bold>10</bold>), 1,5,5-trimethyl-6-acetoxy-cyclohexen-3-one (<bold>11</bold>), and benzyl alcohol (<bold>12</bold>). Compounds <bold>4</bold> and <bold>12</bold> at 1 and <sc>100 μmol L⁻¹,</sc> respectively, accelerated seed germination and promoted the development of the protocorm on 1/2 MS medium; compounds <bold>3</bold> and <bold>10</bold> accelerated seed germination at <sc>100 μmol L⁻¹.</sc> This study clarified the active S7 metabolites that induce <italic>D</italic>.<italic> officinale</italic> seed germination for the first time.

Development and application of recombinase polymerase amplification for rapid detection of rice false smut pathogen (Ustilaginoidea virens)

Ustilaginoidea virens (Cke.) Tak. is the causal pathogen of rice false smut, a grain disease which affects both grain quality and yield. In this study, a recombinase polymerase amplification (RPA) assay protocol was developed to detect U. virens directly from rice spikelet. Three pairs of primer were designed from the U. virens GTP binding protein beta subunit (UvG-β1) gene to function both in RPA and in polymerase chain reaction (PCR). The developed RPA assay efficiently detected U. virens from both genomic DNA and mycelial crude extract. A comparison of sensitivity of RPA with PCR and loop-mediated isothermal amplification (LAMP) revealed that the latter two methods failed to detect U. virens from crude extract. The specificity of the RPA primers was evaluated using DNA from other rice pathogens, as well as, by sequencing the RPA amplicons. For validation, the RPA assay was evaluated on 32 different U. virens isolates from eastern and north-eastern India, as well as, on field samples. The developed RPA assay was able to detect U. virens directly from crude extract prepared from infected rice spikelet. The developed RPA assay will be effective for field monitoring of U. virens.

A genetic tool to express long fungal biosynthetic genes

BackgroundSecondary metabolites (SMs) from mushroom-forming fungi (Basidiomycota) and early diverging fungi (EDF) such as Mucoromycota are scarcely investigated. In many cases, production of SMs is induced by unknown stress factors or is accompanied by seasonable developmental changes on fungal morphology. Moreover, many of these fungi are considered as non-culturable under laboratory conditions which impedes investigation into SM. In the post-genomic era, numerous novel SM genes have been identified especially from EDF. As most of them encode multi-module enzymes, these genes are usually long which limits cloning and heterologous expression in traditional hosts.ResultsAn expression system in Aspergillus niger is presented that is suitable for the production of SMs from both Basidiomycota and EDF. The akuB gene was deleted in the expression host A. niger ATNT∆pyrG, resulting in a deficient nonhomologous end-joining repair mechanism which in turn facilitates the targeted gene deletion via homologous recombination. The ∆akuB mutant tLK01 served as a platform to integrate overlapping DNA fragments of long SM genes into the fwnA locus required for the black pigmentation of conidia. This enables an easy discrimination of correct transformants by screening the transformation plates for fawn-colored colonies. Expression of the gene of interest (GOI) is induced dose-dependently by addition of doxycycline and is enhanced by the dual TetON/terrein synthase promoter system (ATNT) from Aspergillus terreus. We show that the 8 kb polyketide synthase gene lpaA from the basidiomycete Laetiporus sulphureus is correctly assembled from five overlapping DNA fragments and laetiporic acids are produced. In a second approach, we expressed the yet uncharacterized > 20 kb nonribosomal peptide synthetase gene calA from the EDF Mortierella alpina. Gene expression and subsequent LC–MS/MS analysis of mycelial extracts revealed the production of the antimycobacterial compound calpinactam. This is the first report on the heterologous production of a full-length SM multidomain enzyme from EDF.ConclusionsThe system allows the assembly, targeted integration and expression of genes of > 20 kb size in A. niger in one single step. The system is suitable for evolutionary distantly related SM genes from both Basidiomycota and EDF. This uncovers new SM resources including genetically intractable or non-culturable fungi.

Effects of UV Stress in Promoting Antioxidant Activities in Fungal Species Тrametes versicolor (L.) Lloyd and Flammulina velutipes (Curtis) Singer

Trametes versicolor and Flammulina velutipes, after submerged cultivation, with intermittent exposure to ultraviolet light (UV), were studied for Total Phenolic content (TP) and Total Proteins (TPR) contents and antioxidant properties against free radicals. The TP and TPR were determined by a spectrophotometric method and Lowry's assay, respectively. Liquid chromatography with mass spectrometry (LC-MS/MS) was used to quantify polyphenols. Different in vitro assays determined the antioxidant activities of the extracts. Mycelia extract from F. velutipes after 14 days and filtrate from T. versicolor after 21 days of incubation gave the highest TP 59.60 ± 0.14 and 50.03 ± 0.66 mg GAE/g d.w., respectively. Mycelia extract from T. versicolor after 28 days of incubation had the highest TPR (183.53 ± 2.84 mg BSAE/g d.w.). The LC-MS/MS analysis indicated that p-hydroxybenzoic and protocatechuic acids are the most abundant. Trametes versicolor filtrate after 14 days and F. velutipes filtrate after 21 days (71.29 ± 0.54% and 73.5 ± 1.81, respectively) had the highest scavenging activity in SOA. Correlation analysis indicated that all extracts' antioxidant (AO) potential strongly correlated with TP (R2 = 0.83-1.0). The data confirmed that stress factors such as UV exposure could stimulate the production of secondary metabolites and natural AOs, especially phenolic acids in test fungi.

Mycelial and secretome proteomic dynamics of L. squarrosulus AF5 in azo dye degradation

Mycoremediation of textile effluents is a practical and eco-friendly approach for mineralizing synthetic dyes. The textile, food processing, chemical, leather, dyestuff, dyeing, and pharmaceutical industries discharge significant amounts of effluents including different types of dyes. One of the largest manufacturers of dyes is the textile industry, and dyes discharged from these industries directly affect the natural ecosystem by entering in to the soil and water. These industries release effluents that are carcinogenic and pose a potential threat to flora and fauna. Distinct fungal species are identified as dye decomposers because these can produce a variety of extracellular enzymes involved in catabolism. The strain isolated from the dye effluents was identified as Lentinus squarrosulus AF5 and had a remarkable ability for decolorization (50–96 %) within 72 h using a dye mix consisting of AB10B, RB5, and RB160 (500 mg L-1. The dynamics of the protein profile was deciphered using gel-free/label-free nLC-MS/MS analysis. Distinct protein profiles in the mycelial extract of Lentinus squarrosulus AF5 were observed following exposure of the strain with dyes mix. About 90 proteins have been depicted by proteomic analysis wherein 10 proteins appear to be differentially expressed during dye treated conditions. Secretome proteome had denoted 13 proteins under similar conditions. Notable levels of manganese peroxidase (258.84 ± 0.001 U/ml) along with relatively lower levels of lignin peroxidases (194.98 ± 0.002 U/ml) and laccase (134.33 ± 0.007 U/ml) were detected during dye treated condition, suggesting thereby the extracellular peroxidases displaying a pivotal role in decolorization process. The UV-Visible, FTIR spectroscopic along with HPLC and LC-QTOF-MS analyses of the dye supernatants following incubation with Lentinus squarrosulus AF5 had indicated the degradation of the dyes. These observations suggest Lentinus squarrosulus AF5 to be a potential strain for the catabolism of azo dyes.