Abstract

In southwest China, Guizhou province has a well-known fame for its prolific biodiversity of medicinal plants. For solving the problems of plant shortage, environmental disruption and obtaining novel structural as well as bio-active natural products, we have been studying the fungal endophytes and their secondary metabolites from medicinal plants in Guizhou province for a number of years. We isolated over five thousand fungal strains from medicinal plants including Artemisia carvifolia, Artemisia japonica, Blumea balsamifera, Camptotheca acuminate, Dendrobium orchids, Ginkgo biloba, Nothapodytes pittosporoides, Reineckia carnea, Taxus brevifolia. Dozens even hundreds of different fungal isolates were obtained from each plant species. Most of the fungal endophytes belong to Ascomycota which mainly distribute in Pezizomycete, Dothediomycete and Sordariomycete. A few of them are classified into Basidiomycota and Zygomycota. Fungal endophyte taxa were subjected to vary kinds of factors, for example, the age of host, organ, humidity of sample site, altitude, season, surrounding plant, extent of environmental contamination and so on. We combined bio-activity analysis in vitro, chemical composition identification and gene detection in fungal endophyte to screen and evaluate 1003 fungal strains. Fifty-six of them showed anti-inflammatory, antitumor, antioxidant, anti-pathogen and P-gp inhibitory bio-activity to different extent. At present, we accomplished the study for secondary metabolites of ten fungal strains that possess high bio-activity. More than 200 bio-active compounds were isolated. Ten of them have new structures. However, there is a long way to produce bio-active compounds in large scale because of the low production. Therefore, it is necessary to improve cultivation methods or alter inner gene in fungal endophyte by genetic engineering for achieving novel chemical structure as potential new drug.

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Ganoderma lucidum strains are able to produce biologically active polysaccharides. It is widely known that stresses are inducing alteration of metabolome. However, the salt stress effect on G. lucidum has not been studied yet. The purpose of this work was to assess the impact of sodium chloride stress on the mycelia G. lucidum growth and polysaccharide production. We have examined several strains from our samples for the main work objective determination. An identification of ITS rDNA sequences has been conducted with a purpose of specifying the taxonomic position of four strains. For further work we selected G. lucidum strains capable of forming an alkali-soluble and water-soluble polysaccharides with high antitumor activity and capability to cytokines induction. Preclinical trials of an alkali-soluble highly branched xylomannan are in progress now.NaCl effect on the growth of G. lucidum was studied during its introduction into the dense and liquid nutrient culture medium in an amount of 0.5, 1.0, 1.5 and 2.0%. The sharp reduction of fungus colony diameter was observed in medium containing 1.0% or more of sodium chloride. When we added 2.0% NaCl to the dense medium G. lucidum colonies diameter was 21.4% of that of the control. IC50 NaCl estimated concentration was 1.45%.When concentration of chloride in the liquid medium was from 0.5 to 2.0%, we observed the gradual decrease G. lucidum biomass from 13.3 to 76.5%, respectively. The process was linear. Estimated concentration of the IC50 NaCl for submerged cultivation was 1.48%. In medium containing 12.5 g/l of sodium chloride and above, we observed the formation of brown pigment.The total polysaccharides in G. lucidum mycelium have been gradually reduced to 84.6% with 1,0% NaCl, and then have been increased to 171, 6% with 2,0% NaCl to the control. We assessed the changes of the content of monosaccharides as part of studied polysaccharides. Increase of sodium chloride concentrations in the liquid medium to 1.0% has been accompanied by an increase of the total protein in the mycelium to 167.2% compared with that in the control. Further increase of NaCl in the liquid medium to 2.0% was accompanied by a gradual decrease in protein up to 137.7 % to the benchmark. We used the method of light micrsoscopy and method of scanning electron microscopy to reveal micromorphological changes of immersed G. lucidum mycelium when it is grown in medium containing NaCl. In particular, we observed «ball-like» structures, an increase of chlamydospores and hyphae crystals with increase of sodium chloride concentration.

Abstract

Antarctica is one of the most suitable places for the bioprospecting of psychrotrophic fungi. The aim of this study was to investigate the diversity of filamentous fungi from 25 De Mayo Island, Antarctica and their ability to produce extracellular hydrolytic enzymes at low temperature. A total of 51 fungi isolates were obtained from 31 different samples. We identified twelve different genera, seven taxa belonged to the Ascomycota phylum (Cadophora, Helotiales, Monographella, Oidodendron, Penicillium, Phialocephala, Phialophora, Phoma and Pseudogymnoascus), one taxa to the Basidiomycota phylum (Irpex) and two taxa to the Mucoromycota phylum (Mortierella and Mucor). Some taxa not previously reported in Antarctica, as Monographella lycopodina, Mucor zonatus and Penicillium kojigenum, were identify. Nine isolates could not be identified to genus level, and could be representing novel species. Most of the fungi were psychrotrophic (76.5%) rather than psychrophilc. Nevertheless, only five isolates were able to grow at 35ºC, and the optimal temperature for growth was 15ºC for 65% of the fungal isolates. Results from enzymes production (amylase, cellulase, xylanase, lipase, esterase, laccase, protease) at low temperatures revealed that the Antarctic environment contains metabolically diverse cultivable fungi, which represent potential tools for biotechnological applications in cold regions.

Abstract

Soil degradation is a serious issue in the European Union, causing the loss of more than 340,000 areas. LIFE BIOREST (LIFE15 ENV/IT/000396, www.lifebiorest.com) is a UE funded project in the framework of the LIFE Project, aimed to treat a soil contaminated by PHAs, BTEX and alkanes. This site (about 80,000 m² wide) is located in Italy (Fidenza, Emilia Romagna) and has a long history of industrial exploitation. The project aimed to optimize a biomerediation method where the transformation made by consortia of fungi and bacteria is finalized by the final step of re-vegetation. The first phase of the project is indeed focused to characterize the microbial community that naturally populate this extreme environment and isolate those microbes capable of growing in the presence of pollutants as sole C source. The best performing strains will be used to set up consortia working in microcosms and mesocosms before up-scaling the process at in-situ level (biopiles). A solid screening and a liquid enrichment using few selected contaminants (naphthalene, pyrene, phenanthrene, benzene, alkanes and oil extracted from the soil) were carried out to identify the strains with the best adaptation and degradation skills. Despite the strong contamination, microbial communities was consistently developed: more than 220 fungi belonging to 70 species have been identified. Most of the fungal strains belonged to Ascomycetes (mainly to the genera Aspergillus, Cladosporium, Fusarium and Scedosporium ) even though almost 20 Basidiomycetes were also isolated. A further screening was based on an innovative miniaturized approach in 96 multiwell plates in order to evaluate the growth rate of each strain in the presence of 6 contaminants. During the 3 weeks experiments, several strains were capable of growing on the pollutants (at 200 ppm and 1% v/v) as much as positive controls with glucose, highlighting their capability to exploit complex source of nourishment as far as simple and bioavailable ones. Since the bioavailability of organic pollutants in soil is a recognized issue that often limit the efficiency of bioremediation approaches, strains were also screened for their capability to produce biosurfactants. Some fungi were found capable of producing extracellular broths with both emulsifier and biosurfactants activity. Almost 30 fungi and 30 bacteria have been selected and will be tested in micro and mesocosms singly and in consortia with selected bacteria in order to evaluate also their capability to grow and colonize the contaminated soil, and ultimately decontaminate it within 3 months treatment. According to the degradation skills, one consortia was selected for biopile trials. Fungi demonstrated that they could be successfully coupled with bacteria and plants to sensibly reduce the environment hazard of contaminated land, ultimately restoring their ecological functions.

Abstract

Benzene, toluene, ethylbenzene, and the isomers of xylene (BTEX) are volatile anthropogenic pollutants derived from petroleum products that cause harmful effects in humans and other organisms. Fungi were isolated from a 35-ha hypersaline coastal lagoon, Punta Cuchara Natural Reserve, Ponce, Puerto Rico, which is an important nursery for marine species and serves as an avian refuge. Coastal lagoons frequently are contaminated with BTEX. A total of 25 culture-dependent fungal species from the lagoon included the following: Aspergillus sp., Penicillium sp., Cladosporium sp., Trichoderma sp. Fusarium sp., Curvularia sp., Chaetomium sp., Blastoschizomyces capitatus, Candida albicans, C. glabrata, C. rugosa, C. parapsilosis, C. tropicalis, C. zeylanoides, Cryptococcus neoformans, C. albidus, and C. uniguttulatus, Geotrichum sp., Kluyveromyces sp., Prototheca zopfii, Rhodotorula minuta, R. mucilaginosa, Saccharomyces cerevisiae, Trichosporon cutaneum, and Yarrowia lipolytica. A static culture system, each consisting of a serum bottle capped with Teflon Mininert™ valves was used in each of three successive trials to determine degradation of BTEX by each fungi species. An aqueous mineral medium stock solution was prepared with NaNO3 (3.0 g/L), KCl (0.5 g/L), MgSO4 (0.5 g/L), FeSO4 (0.01 g/L), and K2HPO4 (1.0 g/L). Each fungal species was inoculated at a concentration of 1 x 104 yeast cells/mL into the static culture system with a mixture of 118.75 mL of the mineral stock solution and 6.25 mL of BTEX. The BTEX was the only source of energy and carbon. The inoculation was incubated for 150 hr at 25oC. HPLC-DAD method was used to determine the biodegradation of the BTEX in each system by each fungus. The majority of the fungi species degraded the BTEX completely, although traces of BTEX were detected for some species. Thus, funguses species appear to serve as biodegradation organisms in hypersaline lagoons, many of which could be contaminated from petroleum spills.

Abstract

Water-damaged housing has been associated with a number of negative health outcomes, principally respiratory disease and asthma. Much of what we know about fungi associated with water-damaged buildings has been gleaned from culture-based and immunochemical methods. A limited number of studies have used high-throughput sequencing technologies to assess the impact of water-damage on microbial communities in residential buildings. In this study we used amplicon sequencing and quantitative-PCR to evaluate fungal communities in a condemned public housing building in Richmond, CA, before it’s residents were relocated. We recruited 21 households to participate in this study and characterized their apartments as either a unit with visible mold or no visible mold. We collected settled dust from bathrooms, kitchens, bedrooms and living rooms from units with and without visible mold, and from the outdoors. We recovered 5,333 OTUs from 92 samples. We found that fungal biomass was greater outdoors compared to indoors, yet there was no significant difference in fungal biomass in units with visible mold and no visible mold. Fungal richness was significantly reduced in units with visible mold compared to units with no visible mold and the outdoors. We also found that units with visible mold harbored fungal communities distinct from units with no visible mold or from the outdoors. Units with visible mold were dominated by taxa within the classes Eurotiomycetes, Microbotryomycetes, Saccharomycetes, and Wallemiomycetes. A number of the OTUs recovered in significantly greater abundance from units with visible mold, such as Alternaria alternata, Cladosporium sphaerospermum, Rhodotorula mucilaginosa, and Wallemia muriae, have previously been reported with water-damaged building materials. This study demonstrates that long-term negligence and poor building maintenance in low-income public housing impacts not only the human inhabitants, but also the fungi.

Abstract

The widespread development of resistance to antibiotics amongst bacteria pathogenic to humans has led to interest in finding new antimicrobials. We hypothesised that Macrofungi are a likely source of novel antibiotics since their mode of nutrition makes them vulnerable to competition. Amongst the macrofungi, saprotrophs export enzymes to digest macromolecules and must compete with bacteria for the breakdown products and ectomycorrhizal species need to protect the zone of nutrient exchange with plant roots. Accordingly, the production of antimicrobial compounds to control competition for nutrients would provide a competitive advantage to macrofungi. To test our hypothesis, extracts of 170 species have been screened for antibiotics including compounds that block the bacterial efflux pumps that expel antibiotics and compounds that impede the formation of bacterial biofilms. We have screened fruiting bodies and, where possible, also cultures for compounds active against a panel of sixteen bacterial species, including the ESKAPE group of pathogens that cause troublesome hospital acquired infections. We found that a substantial proportion of Australian macrofungi screened do produce antibacterial compounds, including some that inhibit the formation of biofilms or a Staphylococcus aureus efflux pump. One of the new antibacterial compounds identified is non-toxic to human tissue cultures and is amenable to synthesis, offering the possibility of a new family of antibiotics

Abstract

Antibiotic resistance and neurodegenerative diseases are two major medical issues we have to face and which will become even more serious in the future. In order to cope with them and to develop improved therapies new classes of bioactive natural products with different modes of action are needed urgently. Hericium spp. of the phylum basidiomycota are among the most praised medicinal and edible mushrooms, and they have been known to produce secondary metabolites, such as hericenones and erinacines, which were isolated from the fruiting bodies and cultured mycelium, respectively. Many of these compounds were found to promote nerve growth factor (NGF) biosynthesis. Recently, corallocin A-C were isolated from basidiomes of the species Hericium coralloides and were the first members of this compound family that were found to be able to modulate both, NGF and brain-derived neurotrophic factor (BDNF) production. This prompted us to extend our studies to the metabolites from cultures of Hericium species, where metabolite profiles of a strain of the Lion’s Mane mushroom (Hericium erinaceus) and a strain of the rare species, Hericium flagellum (synonym H. alpestre) were examined. Highly similar metabolites were observed in both strains, with cyathane diterpenoids being the predominant ones. Seven metabolites obtained from H. erinaceus and H. flagellum were evaluated regarding their neurotrophin inducing effects. Although none of the tested compounds showed intrinsic neurothrophic activity, erinacines A, B, C, CJ14.258 and the new derivative Z1 clearly enhanced the neurotrophin production in astrocytic cells. Moreover, for the first time we observed a promoting effect of cyathane diterpenoid derivatives on BDNF expression. As they are able to stimulate the transcription of both neurotrophins, they may act upstream on a common molecular target of both pathways or via a third independent pathway.

Abstract

Dementia is a neurodegenerative disease that has become one of the major issues in the 21th century. However, till date there is no specific treatment that can cure or prevent dementia from occurring in humans. Commercial drugs for dementia can only reduce the symptoms of the disease and even worse long term consumptions might pose adverse effects to other bodily functions. Therefore, this study aims to investigate the potential of Hericium erinaceus extracts to stimulate neurite outgrowth in PC12 cells. Briefly, fresh H. erinaceus was cut to cube, freeze dried and extracted with 20% w/v of 95% ethanol and overnight macerated with deionized water followed by 30min hot water extraction respectively. The crude extracts were rotary evaporated and freeze dried before the assay. PC12 cells were treated with 20ug/ml, 40ug/ml, 60 ug/ml and 80ug/ml of the extracts respectively. Neurite bearing score ranged between 2.5-29.6% with the highest score being 50ng/ml NGF treated cells. Pearson’s correlation showed positive correlation between extract concentration and neurite bearing score. Immunofluorescence assay was performed to confirm the neurite outgrowth via staining with rabbit anti-neurofilament 200 polyclonal antibody. The RNAs of the treated cells were also extracted to check the expression level of neurite outgrowth related gene, neuritin via qPCR. This would be the first report of neuritin gene expression following neurite outgrowth induced by mushroom extracts. Further test will be conducted to investigate the effect of co-incubation of H. erinaceus extract with 5ng/ml of NGF. In conclusion, H. erinaceus may be applied as potential health and functional food source in management of neuronal related diseases.

Abstract

Enterovirus infections are amongst the most common infections affecting people worldwide. These viruses cause severe outbreaks especially among children, with symptoms varying from mild to severe including aseptic meningitis, heart muscle damage and paralysis. They have recently been revealed to contribute to chronic diseases, such as type 1 diabetes, as well as with cardiomyopathies and atherosclerosis. Despite efforts of developing anti-virals against enteroviruses that have been going on for years, no vaccines (except for poliovirus) and drugs have made it past the clinical phase and into the market. So far, antiviral effects of extracts obtained from fruiting bodies and/or mycelia in liquid cultures (fermentation) have been described from several mushroom species, of which Ganoderma lucidium is one of the most extensively studied. Crude extracts as well as fractioned compounds including triterpenes and ganoderic acids from G. lucidum have been noted to exhibit inhibitory effects against enterovirus 71 (EV71), herpes simplex virus type 1 and 2 (HSV-1 & 2), as well hepatitis B virus (HBV). Our aim was to evaluate the antiviral effects of crude extracts (water and alcohol extract) from fruiting bodies collected from wild, and liquid fermentation of different strains of G.lucidum isolated from various geographical locations in Finland against enterovirus B group viruses, such as coxsackievirus B3 (CVB3). The Ganoderma extracts and sterile-filtered liquid culture media were incubated for 1 h or shorter periods with the purified virus, and thereafter added on lung carcinoma cells (A549). In addition to infectivity tests, a more detailed investigation to decipher the mechanism of action were performed using confocal microscopy, gradient fractionation and TEM. Our results show a direct inhibitory effect of liquid cultures on enterovirus (CVB3) without cytotoxicity in human cells. We noted a clear difference in the intensity of inhibitory effect between different strains of G. lucidum. In the liquid fermentation, the duration also clearly affected on the results, longer fermentation time giving higher inhibitory effect. These results demonstrate the importance of screening and selection of fungal strains with desired intensity of effect. This is of utmost importance if aiming for production of pharmaceutical products in bioreactors.