Mycelial Extracts Research Articles

Antioxidant and Cytotoxicity Activities of the Fermentation Extract of the Endophytic Fungi from the Marine Biota of Colt Coral

Indonesia has the largest and most biodiverse coral reef in the world. Colt coral has not been studied and explored especially endophytic fungi associated with the coral. Endophytic fungi are highly potential for the production of antioxidant and anticancer compounds. This research aimed to study the antioxidant and cytotoxic activities of fermentation extract from endophytic fungi from colt coral. Filtrate and mycelium extracts were obtained from static and shake fermentations of isolate SKF 15. Antioxidant and cytotoxic assays were conducted by free radical scavenger 1,1-diphenyl-2-picrylhydrazyl (DPPH) and AlamarBlue methods, respectively. The result showed that FD extract provided the highest antioxidant activity with inhibition of 49.36% at 200 ppm of DPPH. Variation of fermentation time (3-21 days) demonstrated the highest activity with inhibition of 66.97% for antioxidant assay (7 days) and 81.13% for cytotoxic assay (3 days). FTIR analysis presented the existence of hydroxyl groups O-H (3452.58 cm-1), C=C groups (1668.43 cm-1); C-O hydroxyl group (1230.58 cm -1), and C-H sp3 groups (2941.44 cm-1). Based on LC-MS analysis, FD extract has a mass of m/z 305.63, [M+H]+, predicted as dihydroquercetin (C15H24O7). Keywords: Antioxidant assay, cytotoxic assay, endophytic fungi, colt coral, DPPH method, AlamarBlue method

Evaluation of nematicidal potential of endophytic fungi associated with healthy plants and GC-MS profiling of metabolites of endophytic Fusarium solani

Endophytic fungi are now recognized as an excellent source of widely occurring natural products. Fungi are wonderful producers of natural products, mostly secondary metabolites. The vital use of these fungi may provide benefit to current demand for novel biomolecules in agriculture, medical and pharmaceutical industries. In this study, endophytic fungi Aspergillus terreus, Cephalosporium sp., Chaetomium sp., Curvularia lunata, Curvularia hawaiiensis, Macrophomina phaseolina, Fusarium solani, Talaromyces assiutensis and Talaromyces trachyspermus isolated from healthy plants and evaluated for nematicidal activity against Meloidogyne javanica, a root-knot nematode. In vitro culture filtrates of these fungi showed strong nematicidal activity by killing juveniles of nematode to varying degree, where F.solani caused 100% mortality after 48 hours. n- Hexane fraction of culture filtrate and n-hexane extract of mycelium of F. solani were subjected to GC-MS analysis, resulted in the identification of several compounds and some are not reported before from this fungus. Metabolites from endophytic F. solani could be used in the development of antimicrobial agents that can be used in medicine or agrochemicals. The use of fungal based products offers a unique opportunity to discover novel therapeutic agents to combat various infectious agents and agricultural pests.

Bio-prospective potential of Pleurotus djamor and Pleurotus florida mycelial extracts towards Gram positive and Gram negative microbial pathogens causing infectious disease

BackgroundThe emergence of resistance to commonly used antibiotics by human infections occurred mostly due to their overuse, that prompted individuals to pursue novel and innovative treatments. The phytochemical characteristics, antibacterial activity, and cytotoxicity of MCF7 cells were evaluated in two Pleurotus spp. mycelial extracts in this work. MethodsPleurotus djamor and Pleurotus florida mycelial extracts from pure cultures were tested for antibacterial activity by a well-diffusion assay and antimicrobial activity against mold fungi was evaluated for biomass inhibition. Mycelial extracts were obtained from dichloromethane extracts and their biophysical characteristics are analyzed by UV–vis spectrum and FTIR analysis. By spraying detection reagents onto TLC plates, the chemicals in dichloromethane extraction of chosen mushroom fungus mycelia were identified. Using the MTT test, the cytotoxic effect of dichloromethane extracts of selected mushroom fungi was evaluated on MCF7 Cell lines. ResultsMycelial extracts of P. djamor and P. florida exhibited significant antimicrobial effect on the bacterial and fungal pathogens tested. Dichloromethane mycelial extracts were obtained using soxhlet extraction which response positive for various phytochemical analysis. Detection of metabolites in thin layer chromatography using spray reagents documented one of few first accounts on flavonoids, anthroquinone and terpenoid compounds in P. djamor and P. florida. P. djamor and P. florida had dose-dependent antiproliferative activity against MCF7 cells, with an inhibitory impact of 55.72% and 64.47% percent at 125 μg/mL, respectively. ConclusionThe study has reported the identification with the potent biological activity of some of the key bioactive components present in DCM extracts from the mycelia of P. djamor and P. florida.

Optimization of Growth Conditions and Biological Activities of Nepalese Ganoderma lucidum Strain Philippine.

Ganoderma lucidum has been extensively studied for its valuable medicinal importance. In this study, the artificial cultivation of G. lucidum strain Philippine in different culture media, including sawdust substrate, was performed and optimized on the Potato Dextrose Agar (PDA) media. Phytochemical, antibacterial, and antioxidant analyses were performed and compared between the ethanol extracts prepared from two different cultures (fruit from synthetic log culture and mycelia from PDA media culture). Both the 200 mg/mL and 100 mg/mL concentrations of extracts inhibited all the tested bacteria, and the results were promising than the corresponding control using antibiotics. The fruit extract showed higher antioxidant potential (150.6 ± 56.92 mg ascorbic acid equivalent/g extract) than mycelial extract (144.28 ± 81.72 mg ascorbic acid equivalent/g extract). The results indicate that fruiting bodies of G. lucidum cultivated in a complex dust medium possess higher antioxidant properties than mycelia culture, which can be further explored for therapeutic applications.

Mitidjospirone, a new spirodioxynaphthalene and GC-MS screening of secondary metabolites produced by strains of Lasiodiplodia mitidjana associated to Citrus sinensis dieback

Mitidjospirone, a new spiridioxynaphthalene, was isolated from the mycelial extract of a strain of Lasiodiplodia mitidjana, a recently described species belonging to the family Botryosphaeriaceae. Its structure was elucidated by extensive spectroscopic analysis and the absolute configuration was determined by electronic circular dichroism (ECD) experiment. Furthermore, several known compounds were identified during the screening of secondary metabolites produced by four strains of L. mitidjana.

Antifungal Activity of an Endophytic Fungus Aspergillus versicolor DYSJ3 from Aphanamixis grandifolia Blume against Colletotrichum musae

An endophytic fungus strain DYSJ3 was isolated from a stem of Aphanamixis grandifolia Blume, which was identified as Aspergillus versicolor based on the morphological characteristics, internal transcribed spacer (ITS) and calmodulin gene sequences analyses. A. versicolor DYSJ3 exhibited strong antagonistic activity against Colletotrichum musae, C. gloeosporioides and Fusarium oxysporum f. sp. cubense with the inhibition rates of 61.9, 51.2 and 55.3% respectively. The antifungal metabolites mainly existed in the mycelium of A. versicolor DYSJ3, and its mycelial crude extract (CE) had broad-spectrum antifungal activities against plant pathogenic fungi. The CE had a good thermal stability, and the inhibition rate of 100 µg/mL CE against C. musae was above 70.0% after disposing at 120 °C for 1 h. Five secondary metabolites were isolated from the CE and identified as averufanin, ergosterol peroxide, versicolorin B, averythrin and sterigmatocystin. Activity evaluation showed versicolorin B exhibited inhibitory effects on the mycelial growth and conidial germination of C. musae, and sterigmatocystin had a weak inhibitory effect on the mycelial growth of C. musae.

The use of gamma irradiation to stimulate bioactive compound synthesis in Inonotus obliquus submerged cultures

Abstract Inonotus obliquus is a parasite on the birch and other trees and is also a well-known medicinal mushroom. Its sterile conk is highly sought for its bioactive compounds such as phenols, polysaccharides, triterpenoids, and steroids. It was traditionally used to treat various gastrointestinal diseases, viral and parasitic infections, to counteract the progression of cancers, and to stimulate the immune system. We used acute gamma irradiation, followed by short-term submerged cultivation, as an oxidative stress inducer to enhance the synthesis of mycelial metabolites. The 300 Gy and 400 Gy doses showed the best results across the whole experimental design. Each assayed criterion had a different corresponding optimal stimulation dose. In one experiment, sublethal doses of irradiation triggered the dry weight of the cultured mycelium to increase by 19.764%. The free radical scavenging potential of the mycelium extracts increased by 79.83%. The total phenolic content of mycelium extracts and culture broth increased by 55.7% and 62.987%, respectively. The total flavonoid and sinapinic acid content of the broth increased by 934.678% and 590.395%, respectively. As such, gamma irradiation pre-treatment of the mycelial inoculum proved an interesting, economically and environmentally effective tool for stimulating secondary metabolite synthesis in submerged mycelium cultures.

Identification of Substances Produced by Cercospora brachiata in Absence of Light and Evaluation of Antibacterial Activity.

Cercospora brachiata is a phytopathogenic fungus. To know more about the metabolites produced by this fungus, the objective of this work was to identify, isolate and characterize substances present in extracts of the growth broth and mycelium, using gas chromatography with mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR). It was also objective to evaluate the antibacterial activity of the extracts. Among the compounds identified, fatty acids, esters, and steroids can be highlighted. The main compounds identified are 9-hexadecenoic, hexadecenoic, oleic, octadecanoic, lauric, myristic, palmitic, doceno-13-enoic, stearic, linoleic, and nonadecanoic acids present in almost all extracts. For the antibacterial activity, the broth microdilution method was used. The ethyl acetate extract of the mycelium presented inhibitory concentrations (MICs) against the bacterium Actinomyces naeslundii (100 μg mL−1) and Streptococcus sanguinis (200 μg mL−1). Finally, two steroids were isolated and identified in the hexane extract of mycelium: ergosta-6,22-dien-3β,5α,8α-triol and brassicasterol.

Antimicrobial and cytotoxic activity of fungal mycelial extracts from aquatic environments in the Amazon

Fungi are a prolific source of biologically active metabolites, including a wide range of clinically important drugs. Therefore, this study aims to evaluate the antimicrobial and cytotoxic activity of secondary metabolites extracted from fungal mycelia isolated from freshwater samples in the state of Amazonas. Mycelial extracts from 12 fungal were used, extracted with MeOH/AcOEt (1:1) according to the criteria established by Souza et al. (2004). For antimicrobial activity, the extracts were tested against the pathogens Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Enterococcus feacalis, Candida albicans and C. tropicalis. To identify the minimum inhibitory dosage (MID) the microdilution method was used. To perform the cytotoxicity assay, the VERO strain (ATCC® CCL-81TM) was used. The assays were determined by the Alamar Blue method according to Ahmed et al. (1994). The tested extracts did not show antibacterial activity. Five extracts (41.7%), obtained from the fungi Aspergillus - 1283, Chrysoporther - 1169, Diaporther – 1203, Fusarium – 1085, and Trichoderma, showed antifungal activity against C. albicans. Diaporther extract (8.3%) - 1203 was active against C. tropicalis. In the cytotoxicity assay, 58.3% of the evaluated extracts showed no significant toxic effect. Five extracts, Cladosporium - 1135, Chrysoporther - 1169, Cytospora - 1098, Fusarium - 1085, and Talaromyces - 1244, showed cytotoxic potential, exhibiting viability lower than 70%. The results obtained suggest that mycelial extracts of fungi isolated from water samples from the Amazon region have potential against yeasts of medical interest. Only two of the active extracts were revealed potentially cytotoxic.

Functionalization with bio-molecules derived from oyster mushroom (Pleurotus florida) diminished the antibacterial potential of the mycogenic metal oxide nanoparticles (nps)

Mushrooms are mostly decomposers and possess the ability to digest food extracellularly by secreting specific enzymes. These enzymes and other bio-molecules present in the mycelium extracts of the P. florida have been used to produce zinc, copper and iron NPs extracellularly using metal salts as precursors. This work includes synthesis of metal oxide NPs by use of different concentrations (0.1to 0.9 mM) of three metal salts viz., zinc, copper and iron which were formed by incubating the salts with mycelium extracts of P. florida up to 96 hours under shaking conditions at 25±2°C in BOD incubator. The synthesis of the NPs was identified by performing the UV-Vis spectroscopy analysis at regular intervals to observe the time-dependent NPs synthesis. The visual color change in the reaction mixture was recorded and development of white, green and brown colors in zinc, copper andiron salt precursor containing sols respectively was recorded. The shape and formation of NPs was identified through TEM analysis. Incubation of the ferric chloride salt precursor with mycelial extract produced cubic shaped NPs. The antibacterial activity studies of the developed NPs using different concentrations (0.1 to 0.9 mM) along with the antibiotic standards (penicillin and gentamycin at the rate of 200 and 20 mg L-1 respectively) were performed against both gram positive and gram-negative bacteria. The results revealed formation of no inhibition zone by the biogenic NPs as compared to inhibition zones formed by the standard antibiotics (maximum 17 mm for E . coli in gentamycin).

Unveiling Biological Activities of Marine Fungi: The Effect of Sea Salt

There is an urgent need for new substances to overcome current challenges in the health sciences. Marine fungi are known producers of numerous compounds, but the manipulation of growth conditions for optimal compound production can be laborious and time-consuming. In Portugal, despite its very long coastline, there are only a few studies on marine fungi. From a collection of Portuguese marine fungi, we screened for antimicrobial, antioxidant, enzymatic, and cytotoxic activities. Mycelia aqueous extracts, obtained by high pressure-assisted extraction, and methanolic extracts of culture media showed high antioxidant, antimicrobial, and cytotoxic activities. The mycelium extracts of Cladosporium rubrum showed higher antioxidant potential compared to extracts from other fungi. Mycelia and culture media extracts of Aspergillus affinis and Penicillium lusitanum inhibited the growth of Staphylococcus aureus, Kocuria rhizophila, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, including multiresistant strains. Penicillium lusitanum and Trichoderma aestuarinum inhibited the growth of clinical strains of Candida albicans, C. glabrata, C. parapsilosis, and C. tropicalis. All extracts from culture media were cytotoxic to Vero cells. Sea salt induced alterations in the mycelium’s chemical composition, leading to different activity profiles.

Diversity of Marine Macro-Algicolous Endophytic Fungi and Cytotoxic Potential of Biscogniauxia petrensis Metabolites Against Cancer Cell Lines.

Hypersaline environments are known to support diverse fungal species from various orders. The production of secondary metabolites is one of the strategies that fungi adopt to thrive under such extreme environments, bringing up the stress tolerance response. Some such unique secondary metabolites also exhibit clinical significance. The increasing prevalence of drug resistance in cancer therapy demands further exploration of these novel bioactive compounds as cancer therapeutics. In the present study, a total of 31 endophytic fungi harboring inside red, green, and brown marine algae have been isolated and identified. The maximum likelihood analysis and diversity indices of fungal endophytes revealed the phylogenetic relationship and species richness. The genus Aspergillus was found to be the dominating fungus, followed by Cladosporium spp. All the isolated endophytic fungal extracts were tested for their cytotoxicity against HeLa and A431 cancer cell lines. Nine isolates were further analyzed for their cytotoxic activity from the culture filtrate and mycelia extract. Among these isolates, Biscogniauxia petrensis showed potential cytotoxicity with CC50 values of 18.04 and 24.85 μg/ml against HeLa and A431 cells, respectively. Furthermore, the media and solvent extraction optimization revealed the highest cytotoxic active compounds in ethyl acetate extract from the potato dextrose yeast extract broth medium. The compound-induced cell death via apoptosis was 50–60 and 45% when assayed using propidium iodide-live/dead and loss of mitochondrial membrane potential assay, respectively, in HeLa cells. Four bioactive fractions (bioassay-based) were obtained and analyzed using chromatography and spectroscopy. This study reports, for the first time, the cytotoxic activity of an endophytic fungal community that was isolated from marine macro-algae in the Rameswaram coastal region of Tamil Nadu, India. In addition, B. petrensis is a prominent apoptotic agent, which can be used in pharmaceutical applications as a therapeutic.

Crude peptides extracted from dry mycelium of Penicillium chrysogenum serve as a micro-associated molecular pattern to induce systemic resistance against tobacco mosaic virus in tobacco

Control of viral diseases using systemic resistance-inducing microbes and microbial metabolites/molecules is an established alternative strategy for inhibiting plant viruses. However, the mechanisms whereby virus infection is inhibited and restricted in plants expressing systemic resistance remains poorly understood. We have previously reported that an aqueous extract of the dry mycelium of Penicillium chrysogenum (DMP) can be used to protect tobacco plants against Tobacco mosaic virus (TMV). In this study, we investigated the effect of crude peptides derived from DMP (PDMP) in protecting Nicotiana glutinosa against TMV. The number and diameter of TMV lesions in plants pre-treated with PDMP were fewer and smaller, respectively, than those in control plants. The elevated expressions of PAL, PTI5, PR-1a, NPR1, PR-1b, and PDF1.2 detected in the induced and systemic leaves following PDMP treatment indicated that the PDMP induced basal and systemic resistance in N. glutinosa. Callose deposition and TMV-N gene-triggered hypersensitive response (HR) at the site of the TMV infection were identified as key factors restricting the movement of viruses. The levels of N gene transcripts, as well as those of the TMV-N gene-triggered HR-related genes HSR203J, AOXa, SIPK, and ZFT1 increased more rapidly after being challenged with TMV in N. glutinosa plants pre-treated with PDMP compared with the control plants. However, we detected no significant difference between PDMP-treated and control plants with respect to TMV-induced callose deposition, indicating that PDMP enhances the resistance of N. glutinosa against TMV by accelerating TMV-N gene-triggered-HR rather than by priming callose deposition.

Potent protective effects of polysaccharide peptide on vascular endothelium and cardiac remodelling in post myocardial infarction patients: A prospective double-blind randomized controlled trial

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Indonesia"s Ministry of Research and Technology/National Research and Innovation Agency grants Background Inflammation and oxidative stress contribute to endothelial dysfunction leading to progression of atherosclerosis and myocardial infarction (MI). Purpose To evaluate the effect of polysaccharide peptide of Ganoderma lucidum mycelium extracts (β-1,3/1,6-D-Glucan) as protective vascular endothelium and cardiac reverse remodelling in post MI. Methods This is a prospective, randomized double-blind clinical trial in 45 post MI patients. Treated group (TG) was given 3 divided dose of 540 mg β-1,3/1,6-D-Glucan daily for 90 days, and control group (CG) received placebo. Variable measured were EPC,CEC,IL-1,IL-6,TNF-α,hsCRP,NO,SOD,MDA and echocardiography parameters. Results β-1,3/1,6-D-Glucan administration significantly increased EPCs (P = 0.000), but not significantly reduced CECs (P = 0.069). Significant suppression was found in IL-1, IL-6 and TNF-α (all P < 0.05), but not in hsCRP (P = 0.064). TG exhibited significant elevation of NO (P = 0.02) and SOD (P = 0.023), and reduction of MDA (P = 0.001). TG revealed significant improvement of LVEF, LVMI, LVIDd, RWT, RWMA and LV diastolic dysfunction (all P < 0.05); but not in FS and TAPSE (P = 0.24, P = 0.20; respectively). Conclusions Polysaccharide peptide exhibits potent protective vascular endothelium and cardiac reverse remodelling in post MI patients, hence promising as add-on medication on top of standard medical regiment. Result comparison between study groupsVariableMeanPretestMean Difference Post-testP valuePolysaccharide peptide (β-1,3/1,6-D-Glucan)PlaceboVascular Endothelium BiomarkersCECsEPCs87.99 ± 76.7398.32 ± 44.52-48.01 ± 64.0714.90 ± 24.05-13.84 ± 58.67-17.65 ± 27.870.0690.000Oxidative Stress MarkersSODMDANitric Oxide2.93 ± 0.5419.68 ± 9.9737.98 ± 14.440.26 ± 0.74-5.52 ± 10.756.04 ± 9.27-0.23 ± 0.685.75 ± 10.29-0.62 ± 3.300.0230.0010.02Proinflammatory BiomarkershsCRPTNF-αIL-1IL-62512.90 ± 1332.11348.58 ± 85.0392.14 ± 29.62259.40 ± 125.52-6.78 ± 1099.60-44.98 ± 100.94-15.77 ± 27.06-29.63 ± 104.30-1.42 ± 795.35-1.34 ± 104.893.42 ± 33.1475.77 ± 130.830.0640.0060.0420.005Echocardiographic ParametersLVEFFSLVMITAPSELVIDdRWT57.20 ± 11.3930.85 ± 7.07114.07 ± 59.612.14 ± 0.354.86 ± 0.820.54 ± 0.352.30 ± 9.770.73 ± 7.59-4.55 ± 14.460.99 ± 0.49-0.49 ± 0.45-0.14 ± 0.29-2.08 ± 3.353.74 ± 9.2711.48 ± 23.94-0.17 ± 0.340.23 ± 0.430.03 ± 0.040.0080.2480.0430.2020.0000.017Abstract Figure. Comparison among pre- and post-treatment

Antimicrobial properties of marine fungi from sponges and brown algae of Mauritius

ABSTRACT Purpose of the study: Marine fungi of Mauritius have been poorly studied. There are numerous reports on the bioactive secondary metabolites that are produced by fungi around the world. Yet, research on the molecular characterisation and the pharmaceutical potential of marine fungi in Mauritius is rather scanty. Method: The samples, which consisted of three sponges Haliclona sp., Iotrochota sp. and Biemna sp. and two brown algae Turbinaria conoides and Sargassum portierianum, were collected in the North of Mauritius during winter. No sporulating structures were observed from the fungal cultures making morphological analysis impossible. The molecular characterisation of the selected isolates was carried out by the amplification of the ITS regions and phylogenetic analysis. The antimicrobial properties were then determined using the disc diffusion and the minimum inhibitory concentration (MIC) assay. Results: Genus level identification was made from molecular data and for some isolates, species-level identification was even possible. Twelve fungi that showed the best antimicrobial properties were identified as Peniophora sp., Aspergillus cristatus, Acremonium sp., Cordyceps memorabilis, Aspergillus ochraceus, Biscogniauxia sp., Aspergillus keratitidis, Exserohilum rostratum, Chromocleista sp., Nigrospora oryzae, Aspergillus flavipes and Mycosphaerella. The lowest MIC result of 0.0098 mg/mL was obtained with Chromocleista sp. mycelium extract against Staphylococcus aureus. The MIC of the mycelium extracts was lower than the broth extracts for most isolates indicating that the antimicrobial compounds are not secreted. Conclusion: Marine fungi from the Mauritian waters have immense potential in the search for natural products against antibiotic-resistant bacteria.

A safety assessment of hot aqueous mycelium extracts from Trametes versicolor and Lepista nuda as a food supplement

Trametes versicolor (L.) Lloyd and Lepista nuda (Bull.) Cooke, known as an edible and medicinal mushroom, draws attention as a source of functional food development. Investigation of these fungal species on blood cultures in terms of human health will supplement the literature. In this study, the biomass of T. versicolor and L. nuda were used to prepare hot aqueous mycelium extracts (HAMEs). HAMEs were evaluated for antioxidant activity and cytotoxicity on human blood cultures (HBCs) in vitro. HBCs were initially incubated with HAMEs in 0.01-2 g/L dose ranges for 1 and 2 days and then analyzed. Firstly, the antioxidant activity on blood serum was determined by using Rel Assay kits with TAC (total antioxidant capacity), TOS (total oxidant status), and OSI (oxidative stress index) values. Secondly, cytotoxicity on lymphocytes was determined using neutral red uptake (NRU) and lactate dehydrogenase (LDH) assays. Doses of 0.05 to 0.5 g/L of T. versicolor and L. nuda increased TAC levels and decreased TOS values. 0.01 and 1 g/L did not change the TOS values but increased it by 2 g/L. In the second case, the dose range 0.01-1 g/L did not offer cytotoxic data. However, 2 g/L caused a 2 % increase in LDH and a 7 % decrease in NRU. As a result, this study presented that any evident signs of toxicity did not accompany T. versicolor and L. nuda HAMEs applications at 0.01-1 g/L dose ranges and 1–2 days on HBCs. The lack of toxicity supports the potential usage of T. versicolor and L. nuda in specified doses as a food or dietary supplement.

Carbohydrate analysis of Mortierella alpina by colorimetry and HPLC-ELSD to reveal accumulation differences of sugar and lipid.

To establish reliable methods for the extraction and quantification of the total carbohydrate and intracellular saccharides from Mortierella alpina and study the changes between carbohydrate and lipid in fermentation process. The extraction of mycelia with HCl following a photometric phenol-sulphuric acid reaction was identified as an optimal method for total carbohydrate analysis in Mortierella alpina, which the extraction efficiency performed 1.1-3.6 fold than other five methods. The total carbohydrate content increased from initial 19.26 to 25.86% during early fermentation process and declined gradually thereafter, while the fatty acid was increasing from 8.47 to 31.03%. For separation and qualitative estimation of intracellular saccharides, the acetonitrile/water freeze-thaw method for extraction and Sugar-Pak I column for separation proved to be possible. With the glucose rapidly decreasing at the beginning of growth, the trehalose accumulated rapidly from 1.63 to 5.04% and then decreased slightly but maintain above 4% of dry biomass. This work established comprehensive carbohydrate extraction and analysis methods of Mortierella alpina and identified the main saccharide in fermentation process which indicated that the accumulation of fatty acids was related to the change of intracellular carbohydrate content.

In vitro EVALUATION OF HYDROALCOHOLIC EXTRACTS OF MYCELIUM, BASIDIOMATA AND SPENT SUBSTRATE OF Pleurotus ostreatus AGAINST Haemonchus contortus

<p><strong>Background. </strong>The edible mushroom <em>Pleurotus ostreatus</em> is a potential alternative for the control of gastrointestinal nematodes due to their nematoxins and secondary metabolites. <strong>Objective. </strong>To evaluate the <em>in vitro</em> anthelmintic activity of hydroalcoholic extracts of mycelium, basidiomata and spent substrate of the edible mushroom <em>P. ostreatus</em> against eggs and unsheathed larvae of <em>Haemonchus contortus</em>. <strong>Methodology. </strong>The mycelium of <em>P. ostreatus</em> was extracted with ethanol–water (70:30) and the basidiomata and the spent substrate with methanol–water (70:30) which were concentrated to obtain the mycelium (MHA), basidiomata (BHA) and spent mushroom substrate extract (SMSE) extracts. The eggs and larvae of <em>H. contortus</em> were exposed to different extracts in 96-well plates. The tests used were the egg-hatch and larval mortality tests. Three exposure times, 24, 48 and 72 h, were considered. The data were analyzed with a factorial design and the comparison of means by Duncan test. <strong>Results. </strong>The percentage anthelmintic effectiveness of the mycelium hydroalcoholic extract (MHA) of <em>P. ostreatus</em> against eggs and larvae of <em>H. contortus</em> were 93 and 96.8% at 12.8 and 240 mg/mL (respectively) after 72 h. Also, activity was observed for SMSE and BHA against eggs, with values of 87.2 and 100%, respectively, at 25.6 mg/mL after 72 h. However, SMSE showed no activity against <em>H. contortus</em> larvae, while the BHA extract showed less than 70% corresponding activity. <strong>Implications.</strong> This was an exploratory study of <em>in vitro</em> tests; the <em>in vivo</em> studies are still needed. <strong>Conclusion. </strong>The MHA extract of <em>P. ostreatus</em> showed anthelmintic activity against the eggs and larvae of <em>H. contortus</em>.</p>

Plant Diseases Containment and Growth Stimulators of Fungal Origin

It is well known that mycopesticides have significant potential for a plant protection. For example, pathogenic fungi can induce nonspecific defence reactions and stimulate growth of non-host plants. The aim of this work is to evaluate the off-target activities of potential mycopesticides which affect germination, growth and development of crops. The strains of Stagonospora cirsii C-163, Calophoma complanata 32.121, Alternaria sonchi S-102, Beauveria caledonica BB16, BSc13Vg18, BSc25Vg18, Beauveria pseudobassiana BCu22, Beauveria bassiana T7, BHy1-06 were obtained from the pure cultures collection of the All-Russian Institute of Plant Protection. The most significant leaf spot containment (near 70%) was shown by mycelium extract S. cirsii C-163. In the case of leaf rust, the disease containment effect was lower and was observed for mycelium-based suspension of S. cirsii (near 35%). The maximum growth-promoting effect was observed for mycelium-based suspension of C. complanata. Multivariate analysis data showed that the species of B. caledonica and C. complanata are able to inhibit the development of wheat diseases and promote plant growth. Thus, in our opinion, the screening of producer strains should include the assessment of some nonspecific effects on the development and cultivation of plants.

Characterization and Toxicity of Crude Toxins Produced by Cordyceps fumosorosea against Bemisia tabaci (Gennadius) and Aphis craccivora (Koch).

Cordyceps fumosorosea, an insect pathogenic fungus, produces different toxins/secondary metabolites which can act as pest control agents. This study reports the extraction and characterization of crude mycelial extracts of C. fumosorosea isolate SP502 along with their bio-efficacy against Bemisia tabaci and Aphis craccivora. Fourier transform infrared spectroscopy, liquid chromatography, mass spectrometery and nuclear magnetic resonance analysis of C. fumosorosea isolate SP502 extracts showed the presence of five major compounds—Trichodermin, 5-Methylmellein, Brevianamide F, Enniatin and Beauvericin—which all may potentially be involved in insecticidal activity. The HPLC analysis of C. fumosorosea mycelial extracts and Beauvericin standard showed similar chromatographic peaks, with the content of Beauvericin in the crude toxin being calculated as 0.66 mg/ml. The median lethal concentrations of C. fumosorosea mycelial extracts towards first, second, third and fourth instar nymphs of A. craccivora were 46.35, 54.55, 68.94, and 81.92 µg/mL, respectively. The median lethal concentrations of C. fumosorosea mycelial extracts towards first, second, third and fourth instar nymphs of B. tabaci were 62.67, 72.84, 77.40, and 94.40 µg/mL, respectively. Our results demonstrate that bioactive compounds produced by C. fumosorosea isolate SP502 have insecticidal properties and could, therefore, be developed into biopesticides for the management of B. tabaci and A. craccivora.