Antimicrobial Activity of Fomitopsis Officinalis (Vill.) Bondartsev & Singer in Pure Culture

Background. According to the World Health Organization antibiotic resistance is among the top ten threats to human health, food safety and development. Today antibiotic resistance has reached alarmingly high levels all over the world. Meanwhile, the increase in the synthetic drugs' production has led to the pathogenic mycobiota's rapid adaptation to the created chemicals, which have a narrow focus of application. That is why in modern biotechnology and pharmacology much attention is paid to natural producers of biologically active compounds, in particular – to xylotrophic fungi. It has been experimentally proven that the xylotrophic macromycete Fomitopsis officinalis or tinder fungus can be considered to be a promising producer of pharmacological substances with a broad spectrum of action. Studies of active metabolites, contained in the mycelial mass, culture fluid of the medicinal xylotrophic macromycete F. officinalis, and determination of their biological action remain relevant. Objective. The objective was to determine the antimicrobial activity of culture fluid and mycelial mass of F. officinalis different strains from the mushrooms collection (IBK Mushroom Culture Collection of the M.G. Kholodny Institute of Botany, NAS of Ukraine) against gram-negative and gram-positive bacteria species. Methods. An in vitro study of the antimicrobial activity of ethyl acetate extracts of culture fluid and aqueous-ethyl extracts of mycelial mass for F. officinalis strains IBK-5004, IBK-2497, IBK-2498 against gram-positive Staphylococcus aureus (B-918), Bacillus subtilis (В-901) and gram-negative Escherichia coli (B-906), Bacillus subtilis (B-900), Klebsiella pneumoniae (M-123) bacteria by disc-diffusion method was conducted. Results. High antimicrobial activity of tinder fungus culture fluid and mycelial mass extracts against Staphylococcus aureus was established after the 21st day of cultivation, while on the 28th day the zone of growth retardation was maximal (15–25 mm). The highest indices were recorded in F. officinalis IBK-5004 (20–25 mm) and IBK-2498 (20–24 mm) strains. Antimicrobial activity against Klebsiella pneumoniae in culture fluid extracts was manifested on the 21st and 28th days of cultivation. The highest antimicrobial activity against Klebsiella pneumoniae was observed in the culture fluid of the strain F. officinalis IBK-5004, the diameter of the growth retardation zone was 18 mm on the 28th day of cultivation. Mycelial mass's extracts showed moderate activity on the 14th day of cultivation (7-8 mm); maximal activity was recorded on the 28th day (12–22 mm). The most active strain was Fomitopsis officinalis IBK-2498. No antimicrobial activity against test organisms was detected in the following studied strains: Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis. Conclusions. It has been established that the mycelial mass and culture fluid extracts of F. officinalis IBK-5004, IBK-2497, IBK-2498 strains have high antimicrobial activity against Staphylococcus aureus and moderate antimicrobial activity against Klebsiella pneumoniae on the 21st and 28th day of cultivation.

Pathogenicity of Cordyceps javanica (Hypocreales: Cordycipitaceae) to Diaphorina citri (Hemiptera: Liviidae) Adults, with Ultrastructural Observations on the Fungal Infection Process

Entomopathogenic fungi are proposed biological control agents against the Asian citrus psyllid (Diaphorina citri). We quantified the pathogenicity of Cordyceps javanica strain Apopka 97 strain (Cja Apopka 97) (blastospores and conidia) against D. citri using the spray technique. We also used light and scanning electron microscopy to observe the Cja Apopka 97, infection process against D. citri adults at different stages pre- and post-mortem. Our findings demonstrated that psyllid mortality in the blastospore treatment ranged from 8 to 25% within 24–48 h of exposure, compared to 0% in the conidial and control treatments. However, psyllid mortality rate had reached 100% by 7 days after exposure at a concentration of 107 spores/mL, under both fungal treatments compared to the controls (0%). SEM and light microscopy revealed several stages in in the Cja Apopka 97 infection process of D. citri, including spore adherence and germ tube formation within 24 h post-inoculation, penetration pegs and mycelia growth on wings after 72 h, rupturing of cuticle after 96 h and mycelial mass colonizing host body after 144 h. Our study findings provide basic information on the interaction of entomopathogenic fungi with D. citri which will assist in the understanding of the infection process and the potential roles of entomopathogenic fungi in its management.

Detoxification of Molasses and Production of Mycelial Mass and Valuable Metabolites by Morchella Species

Edible wild ascomycetes Morchella rotunda, M. vulgaris and M. conica were cultivated in liquid static and agitated flasks of sucrose and molasses substrates with a C/N ratio of 20 and 25. The impact of four substrates on the production and quality characteristics of morels was examined. Evaluation included determination of the dry mycelial mass, intra-cellular (IPS) and extra-cellular (EPS) polysaccharides, total phenolic (TPC) and antioxidant (TAC) components, proteins, as well as the degree of phenolic content reduction and decolorization of molasses. The influence of agitation conditions was also evaluated. Results showed that substrate consumption, biomass formation and secondary metabolites production were substrate, species, and C/N ratio dependent. Among species, M. conica achieved the maximum biomass (18.16 g/L) and IPS (4.8 g/L) production and significant phenolic reduction (56.6%) and decolorization (26.7%). The maximum EPS (3.94 g/L) was noted by M. rotunda, whereas TPC (32.2 mg/g), TAC (6.0 mg/g) and cellular protein (7.6% w/w) were produced in sufficient amounts. These results strongly support the use of Morchella mushrooms as a biological detoxification agent of molasses in liquid fermentations and indicate their nutritional and medicinal value.

Efficiency of a new Waitea circinata extract against rice pathogens

ABSTRACT Waitea circinata (Warcup & Talbot) is an orchid antagonist mycorrhizal fungus with biocontrol potential against rice pathogens. This study aimed to optimize the extraction method, obtain a new extract and evaluate its efficiency against rice pathogens in vitro and in vivo, as well as to compare it with other extraction methods and W. circinata. The extracts were obtained and screened for in vitro growth inhibition against the pathogens Cochliobolus miyabeanus, Monographella albescens and Sarocladium oryzae, using the following extracts: mycelial, crude, lyophilized and mycelial mass. An additional in vitro assay was performed with the principal rice pathogen (Magnaporthe oryzae), in order to evaluate the conidial germination and appressorium formation. Based on this evaluation, the lyophilized and mycelial mass extracts were tested in vivo against rice blast (M. oryzae) and compared to the W. circinata mycelial suspension, in different application forms (simultaneous and previous). The mycelial mass extract inhibited all the pathogens, and the crude and lyophilized extracts inhibited C. miyabeanus and M. albescens, respectively. The mycelial mass extract inhibited the M. oryzae conidial germination and appressorium formation by 80 %, and the simultaneous and previous applications suppressed the rice blast by 94 %. These results indicate that the new extract can be used to control rice pathogens.

Immunotherapy based on Pythium insidiosum mycelia drives a Th1/Th17 response in mice

Pythium insidiosum is an oomycete that affects mammals, especially humans and horses, causing a difficult-to-treat disease. Typically, surgical interventions associated with antimicrobial therapy, immunotherapy, or both are the preferred treatment choices. PitiumVac® is a therapeutic vaccine prepared from the mycelial mass of P. insidiosum and is used to treat Brazilian equine pythiosis. To better understand how PitiumVac® works, we analyzed the composition of PitiumVac® and the immune response triggered by this immunotherapy in mice. We performed an enzymatic quantification that showed a total glucan content of 21.05% ± 0.94 (α-glucan, 6.37% ± 0.77 and (1,3)(1,6)-β-glucan, 14.68% ± 0.60) and mannose content of 1.39% ± 0.26; the protein content was 0.52 mg ml−1 ± 0.07 mg ml−1. Healthy Swiss mice (n = 3) were subcutaneously preimmunized with one, two, or three shots of PitiumVac®, and immunization promoted a relevant Th1 and Th17 responses compared to nonimmunization of mice. The highest cytokine levels were observed after the third immunization, principally for IFN-γ, IL-17A, IL-6, and IL-10 levels. Results of infected untreated (Pythiosis) and infected treated (Pythiosis + PVAC) mice (n = 3) showed that PitiumVac® reinforces the Th1/Th17 response displayed by untreated mice. The (1,3)(1,6)-β-glucan content can be, at least in part, related to this Th1/Th17 response.

Spent mushroom substrate for a second cultivation cycle of Pleurotus mushrooms and dephenolization of agro-industrial wastewaters

ABSTRACT Spent mushroom substrate (SMS) of Pleurotus ostreatus was supplemented with wheat bran and soybean flour and used as substrate for a new cultivation cycle of the oyster mushrooms Pleurotus ostreatus and Pleurotus pulmonarius. The bioconversion efficiency of mushrooms produced over substrate (BE%) used and the chemical composition of sporophores were evaluated. The concentration of mycelial mass, crude exopolysaccharide content and laccase enzyme activity were also determined at the supplemented SMS before inoculation, at 50% and 100% of colonization stages in the new cultivation and in the final re-utilized SMS. The laccase enzyme was extracted to examine SMS potential for the dephenolization of olive mill and winery wastewaters. Results showed that both Pleurotus species exhibited BE over 185%, demonstrating this bioprocess could represent a promising strategy to convert SMS into nutritional food. Data also indicate the strong positive impact that SMS could have in the solid wastes’ management and agribusiness enhancement.

Researching of features byosinthesis beta-glucans in the micelial mass Aspergillus niger and in non-conditional cereal grains

At the moment, it seems relevant to process mycelial mass as a waste product of citric acid and substandard grain, since there is a problem of recycling production waste. In turn, waste processing makes it possible to isolate a number of unique compounds, such as glucan and chitin. Of interest is the cell wall of micromycetes. Its main structural polysaccharides (chitin and glucan) form a complex called the chitin-glucan complex. Moreover, the content of chitin in micromycetes is higher than in traditional raw materials - shells of marine crustaceans. The mycelial mass of Aspergillus niger micromycete is a promising source of glucan and chitin. Aspergillus niger biomass and substandard cereal grains contain soluble dietary fiber-beta-glucans. They help reduce the risk of cardiovascular disease by lowering cholesterol, control blood glucose, which is very important for patients with diabetes. In addition, because of the ability of beta-glucans to form viscous gels, these chemicals slow down the absorption of sugars. Due to this, there is a decrease in the glycemic index of products. Of greatest interest are glucans of microbial origin. In addition to structural features (more functionally active ?-1,3 / 1,6-bonds), the advantage of beta-glucans derived from biomass is a higher quantitative yield of soluble forms, which are interesting not only in the food industry, but also in medicine . The aim of this work is to study the effect of various carbon sources (raw materials) on the biosynthesis of glucan-containing compounds and their derivatives Aspergillus niger micromycetes. As a result of studies, it was found that the biomass on the corn starch hydrolyzate is the most effective, it contains a high content of beta-glucans.

Upgrading Grape Pomace through Pleurotus spp. Cultivation for the Production of Enzymes and Fruiting Bodies

Grape pomace, a by-product derived from winery industries, was used as fermentation media for the production of added-value products through the cultivation of two Pleurotus species. Solid-state (SSF), semiliquid (SLF), and submerged (SmF) fermentations were carried out using grape pomace as substrate. The effect of the different fermentations on the consumption of phenolic compounds, the production of mycelial mass and enzymes was evaluated using P. ostreatus and P. pulmonarius. The production of fungal biomass and enzymes was influenced by the fermentation mode. The maximum biomass values of ~0.5 g/g were obtained for both P. pulmonarius and P. ostreatus in SmF. Laccase production was induced in SSF and a maximum activity of 26.247 U/g was determined for P. ostreatus, whereas the highest endoglucanase activity (0.93 U/g) was obtained in the SmF of the same fungi. Analysis of phenolic compounds showed that both strains were able to degrade up to 79% of total phenolic content, regardless the culture conditions. Grape pomace was also evaluated as substrate for mushroom production. P. pulmonarius recorded the highest yield and biological efficiency of 14.4% and 31.4%, respectively. This study showed that mushroom cultivation could upgrade winery by-products towards the production of valuable food products.

Microbial Synthesis and Characterization of Silver Nanoparticles using Alternaria alternata

In this study, extracellular biosynthesis of silver nanoparticles (AgNPs) was carried out using Alternaria alternata. AgNPs were synthesized using vegetative and cell-free filtrate methods. Silver nitrate (AgNO3) solution was reduced with fungal mycelial mass and cell-free filtrate at a ratio of 1:4 (v/v). Further, the effects of biosynthesis parameters (reaction time, reaction temperature, light and dark incubation, and static and agitated conditions) weredetermined. The structural integrity of the synthesized AgNPs was investigated through UV-visible spectrophotometry, X-ray diffraction (XRD) analysis, and transmission electron microscopy (TEM). Parameter optimization revealed that cell-free filtrate AgNP synthesis at 40 °C with constant agitation and incubation in darkness for 120 h using 1 mM AgNO3resulted in the highest absorbance value (at 400 nm). Further, TEM images showed that the synthesized AgNPs were spherical, homogeneous, and well dispersed while the XRD peaks confirmed the purity of the AgNPs obtained. The diameters of the AgNPs were found to range from 7.48 to 12.15 nm. This study identifies Alternaria alternata as a potential candidate for use in the industrial biosynthesis of AgNPs.

Improvement of Soil Properties, Growth of Cucumber and Protection against Fusarium Wilt by Piriformospora indica and Two Industrial Organic Wastes

The current work was focused on characterizing bagasse ash (BA) and press mud (PM) as soil amendments and to study their effect in combination with the endophytic fungus Piriformospora indica on Fusarium wilt (FW) of cucumber caused by Fusarium oxysporum f. sp. cucumerinum (Fo). Whereas BA and PM improved almost all physico-chemical properties of the soil evaluated, seed treatment with P. indica had no such effect. In shake culture in potato dextrose broth (PDB) medium amended with aqueous extracts of BA and PM, alone or in combination, production mycelial mass of Fo was significantly decreased by PM extract, while production mycelial mass of P. indica was highly improved. The colonization rate of cucumber roots by P. indica as determined by microscopy was highly increased by increasing amounts of BA, PM and BA+PM added to the soil. Seed treatment of cucumber with P. indica before plant cultivation in non-amended soil significantly decreased the disease severity of FW and improved plant growth. When seed treated with P. indica was sown into soil amended with BA, PM or the combination of both, the disease severity was even more reduced than after seed treatment with P. indica alone. In this respect, amendment with PM was more effective than with BA, and the combinations were more effective than the single treatments. Hence, there is a scope to integrate PM and BA as soil amendments in combination with P. indica for eco-friendly FW management, improving soil properties and growth of cucumber plants.