Strain Improvement and Strain Maintenance Revisited. The Use of Actinoplanes teichomyceticus ATCC 31121 Protoplasts in the Identification of Candidates for Enhanced Teicoplanin Production

Multicellular cooperation in actinomycetes is a division of labor-based beneficial trait where phenotypically specialized clonal subpopulations, or genetically distinct lineages, perform complementary tasks. The division of labor improves the access to nutrients and optimizes reproductive and vegetative tasks while reducing the costly production of secondary metabolites and/or of secreted enzymes. In this study, we took advantage of the possibility to isolate genetically distinct lineages deriving from the division of labor, for the isolation of heterogeneous teicoplanin producer phenotypes from Actinoplanes teichomyceticus ATCC 31121. In order to efficiently separate phenotypes and associated genomes, we produced and regenerated protoplasts. This approach turned out to be a rapid and effective strain improvement method, as it allowed the identification of those phenotypes in the population that produced higher teicoplanin amounts. Interestingly, a heterogeneous teicoplanin complex productivity pattern was also identified among the clones. This study suggests that strain improvement and strain maintenance should be integrated with the use of protoplasts as a strategy to unravel the hidden industrial potential of vegetative mycelium.

Influence of gibberellin on cultural and growth characteristics of vegetative mycelium of fungi strains of the genus Pleurotus during cultivation on agarized nutrient media

Very slow introduction into the culture of new for Ukraine species of edible fungi is associated primarily with more difficult conditions for the cultivation of these fungi, slow growth of mycelium, difficulties in obtaining a pure culture, sterilization of the substrate and others. Therefore, it is important to study the possibility of using growth stimulants used in crop production in industrial mushroom growing in order to intensify the process of obtaining the mycelium and fruiting bodies of mushrooms. The aim of this experiment was to study the nature of the influence of gibberellin on the cultural and growth characteristics of the vegetative mycelium of strains of the genus Pleurotus, which are classified as medium- and slow-growing on agar nutrient media. The objects of the study were industrial strains of Pleurotus pulmonarius (Fr.) Quél. (strain IBK-230) (medium-growing), Pleurotus eryngii (DC.) Quél. (strains IBK-2011 and IBK-1972) (slow-growing) obtained from the Collection of cap mushrooms of the Institute of Botany named after N.G. Kholodny NAS of Ukraine. The following nutrient media were used in the study: corn agar (CA), crushed sunflower husk agar (SHA), glucose-ammonium (GAM) and glucose-asparagine (GAS) agars. Gibberellin was added to the nutrient media in the following concentrations: 1, 10, 50, 100 mg / dm3. The control media did not contain growth stimulant.Preparation and sterilization of nutrient media, tests for individual enzymes were performed according to conventional methods. 9-day crops grown on corn agar on Petri dishes were used as inoculum. Surface cultivation of the mycelium of the studied strains of mushrooms was performed in a thermostat at a temperature of 26 ± 1 °C.During the cultivation process, the radius of colonies on Petri dishes was measured daily, morphological description was performed, the duration of the lag-phase of mycelial growth on media with different concentrations of gibberellin, the average rate of radial mycelial growth (mm / day) and qualitative color reactions to phenoloxidases (laccases, tyrosinases, peroxidases) was determined. Data processing was performed by methods of mathematical statistics.According to the results of the study of cultural and morphological characteristics of colonies of P. pulmonarius, P. eryngii, it was determined that the applied concentrations of gibberellin do not have a significant effect on the morphological parameters of fungal colonies on agar nutrient media.Taking into account the data of the average daily growth of mycelium of the studied Pleurotus strains on agar nutrient media and the average duration of the lag-phase of growth of fungal colonies, the following can be stated. For P. pulmonarius, a significant positive effect of gibberellin on the lag-phase of growth was observed on the media CA, SHA, GAM – for a stimulant concentration of 1 mg / dm3 (9, 10 and 18%, respectively), CA and SHA – for a concentration of 10 mg / dm3 (9 and 10%, respectively), and on the SHA – for a concentration of 50 mg / dm3 – 20% compared with the control. Gibberellin had the best effect on the duration of the lag-phase of growth of the mycelium of P. eryngii IBK-2011, where the reduction of the lag-phase of growth ranged from 8 to 18% for all concentrations of growth regulator compared to the control.The average rate of radial growth of the mycelium of P. pulmonarius IBK-230 significantly increased compared with the control under the influence of all studied concentrations of gibberellin on the media CA (from 16.8 to 18.8%) and GAM (from 26.3 to 52.6%). The best indicator of linear growth rate was recorded on CA and GAM media with a concentration of gibberellin of 50 mg / dm3. The obtained data from the average radial growth rate of mycelium of slow-growing strains of P. eryngii (IBK-2011, IBK-1972) showed that these strains respond better to the growth stimulator gibberellin: on all nutrient media there was an increase of the growth rate for almost all concentrations of gibberellin from 3.1 to 60%. The most effective concentrations of gibberellin were 10 and 50 mg / dm3 (on the nutrient media of CA and GAM) for all strains of the studied fungi, on GAS – 1, 10 and 50 mg / dm3.The enzymatic activity of the studied strains differed depending on the type of mushrooms, nutrient medium and different concentrations of gibberellin. Thus, for P. pulmonarius no significant difference was found in the manifestation of color reactions to phenoloxidases, except for the enzyme tyrosinase, where the color appeared after 30 minutes on SHA (the concentration of gibberellin – 10 and 50 mg / dm3) and GAM (the concentration of 1 and 10 mg / dm3), on others the corresponding color appeared within 3 hours. The manifestation of color reactions to phenoloxidases for P. eryngii (IBK-2011, IBK-1972) for 30 minutes was observed on CA medium (concentration of gibberellin – 10, 50 and 100 mg / dm3), on SHA – 10 and 50 mg / dm3, on GAM – 1 and 10 mg / dm3, on GAS – 1, 10, 50 and 100 mg / dm3. Thus, slow-growing strains are more responsive to synthetic activity on this growth stimulant.As a result of the experiment it was found that the growth stimulant gibberellin affects the culture and growth characteristics of the vegetative mycelium of industrial strains of the genus Pleurotus, belonging to the medium- and slow-growing – P. pulmonarius (strain ІВК-230), P. eryngii (strains ІВК-2011, ІВК-1972): the lag-phase of mycelial growth decreases (on average from 8 to 18%), the average radial growth rate of mycelium increases from 16.8 to 57.9% for P. pulmonarius, and from 10.5 to 60% for strains P. eryngii, increases enzymatic activity. This growth stimulant can be used in biotechnology of edible fungi to obtain uterine mycelium of slow-growing species, increase the synthesis of biologically active substances by macromycetes, as well as increase the yield of fruiting bodies of mushrooms, which are poorly represented in the consumer market of Ukraine.

STABILITY OF THE STRAINS OF BASIDIOMYCETES DURING STORAGE IN THE COLLECTION

Basidial macromycetes may be a material for the development of new biotechnologies, medical preparations, components of dietary nutrition. Therefore, it is necessary for the highest level of quality for maintenance and identification of mushroom strains in the collection. An important parameter, in this case, is a stability of isolated and described collection strains of basidiomycetes. Stability is one of the key issues of long-term preservation of pure culture collections. For the collection of medicinal basidiomycetes of ONU I.I. Mechnikov, which preserves by the method of periodic reseeding of colonies the strain stability had not been studied yet. The goal of this research is to study the stability of this collection by a growth rate of mushroom colonies and electrophoretic spectra of carboxylesterases after different times of storage of cultures on malt agar. In this research the strains of three age categories (1, 2 and 3 years) of storage on malt-agar medium at temperature 4 ° С for were tested. The radial growth rate of their vegetative mycelium and the spectra of multiple molecular forms of carboxylesterases by the method of vertical electrophoresis in 7% of polyacrylamide gel were investigated. It was established that the stability of the radial growth rate of A. auricula-judae, F. velutipes, G. lucidum the vegetative mycelium after different storage periods is high according to the values of variation coefficients. At the same time, the expression of molecular forms of carboxylesterase showed sufficient variability. Partially conservative molecular forms were detected in some age groups of strains, as well as for individual strains. Thus the growth rate of colonies is a stable indicator and the molecular forms of carboxylesterases of different ages strains are variable.

Ultrastructure of hyphal cells of the dermatophyte Trichophyton tonsurans

Background and Purpose: Trichophyton tonsurans is a widely distributed anthropophilic dermatophyte causing different diseases of skin. In the literature limited data are available about the morphogenesis of vegetative mycelium of T. tonsurans and related anthropophilic dermatophytes. The aim of present study was to describe ultrastructural patterns of development, cellular organellography and septal pore apparatus structure of in vitro growing vegetative mycelium of T. tonsurans. Materials and Methods: Trichophyton tonsurans strain RCPFF 214/898 was grown on solid Czapek’s Agar (CzA) at 28ºС. For investigation of colonies morphology we used methods of light-, scanning and transmission electron microscopy (SEM and TEM). Results: Differences in morphogenesis of aerial and substrate hyphae were revealed. Mitochondrial reticulum and fibrosinous bodies were shown in T. tonsurans for the first time. The septal pore apparatus in hyphal cells of was comprised Woronin bodies and septal pore plugs. Woronin bodies (0.18 μm), located with 1‒4 near the pore, were spherical, membrane-bound, and had a homogeneous, electron-dense content. The cells of aerial and submerged hyphal cells of T. tonsurans contain two nuclei. Conclusion: Mature cells of substrate hyphae appeared more active than comparable cells in the aerial mycelium. During the maturation process, the differences in number and morphology of mitochondria, number of vacuoles, and in the synthesis of different types of storage substances were revealed. Presence of “mitochondrial reticulum” and variable types of storage substances in submerged hyphal cells suggested higher levels of metabolic activity compared to aerial mycelium.

The influence of laser irradiation on the development of vegetative mycelium Pleurotus ostreatus

The article presents the results of the study on the influence of laser irradiation on the development of vegetative mycelium and the period of the occurrence of the corcules of the fruit bodies of Pleurotus ostreatus. It has been established the growth processes of P. ostreatus, can be best stimulated by applying laser irradiation of mycelium with a green spectrum of light for 10 s. According to this mode of exposure, the best increase in the growth rate of mycelium of 38.3% and the appearance of the largest number of rudiments of the fruiting bodies were recorded. Laser irradiation of mycelium for 10 s with red and blue light spectrum increased the growth rate of mycelium from 7.41 to 20.4%, respectively, and the number of rudiments of the fruiting bodies increased by 1.5 to 2 times. Laser irradiation of the mycelium with 5 s, 15 s, and 20 s with red, blue, and green light spectra did not have a significant effect on the growth processes of P. ostreatus. These data open significant prospects for the modification of the existing cultivation technologies, which would increase the economic efficiency of the biotechnological cultivation process of P. ostreatus.

Alginate gel entrapped ectomycorrhizal inoculum promoted growth of cuttings of Eucalyptus clones under nursery conditions

Plant inoculation with ectomycorrhizal fungi (EMF) maximizes the productive potential of forest stands. Thus, the inoculation efficiency of calcium alginate gel entrapped EMF vegetative mycelium was evaluated in a commercial nursery using cuttings of Eucalyptus clones GG100 and GG680. The cuttings were inoculated with Pisolithus microcarpus G. Cunn. (Cooke & Massee), Hysterangium gardneri E. Fisch., and Scleroderma areolatum Ehrenb. The cuttings were cultivated under low phosphate fertilization and compared with uninoculated control treatments with reduced phosphate (low P control) and full phosphate (high P control) fertilization. Pisolithus microcarpus inoculation increased shoot height, root collar diameter, shoot dry mass, total dry mass, and frequency of maximum score for root ball formation of the two clones compared with the low P control treatment. Also, in relation to the low P control treatment, H. gardneri inoculation increased shoot dry mass in GG100 rooted cuttings. Scleroderma areolatum inoculation did not enhance any characteristic of Eucalyptus rooted cuttings. Inoculation of vegetative mycelium with EMF impregnated in calcium alginate gel intensified rooted cutting growth in a commercial Eucalyptus nursery and decreased the phosphate dose required. Based on the comparison of two Eucalyptus clones, efficiency of the inoculants in promoting benefits depends on the fungus and the Eucalyptus clone. Pisolithus microcarpus is most promising for inoculation in Eucalyptus cuttings.

(1→3)-α-d-Glucan from Fruiting Body and Mycelium ofCerrena unicolor(Bull.) Murrill: Structural Characterization and Use as a Novel Inducer of Mutanase

Water-insoluble, alkali-soluble polysaccharide (marked as ASP) was extracted from the vegetative mycelium and fruiting body ofCerrena unicolorstrain. Monosaccharide examination of ASP demonstrated that the isolated biopolymer was composed mainly of glucose, xylose, and mannose monomers. The methylation investigation of studied polymers indicated that (1→3)-linkedα-D-Glcpis the major chain constituent (92.2% for glucans isolated from fruiting body and 90.1% from mycelium).1H NMR, FT-IR, and immunofluorescent labelling determinations confirmed that the polysaccharides isolated from both fruiting body and mycelium ofC. unicolorare (1→3)-α-d-glucans. The obtained (1→3)-α-d-glucans showed differences in viscosity and similar characteristics in optical rotations. (1→3)-α-d-Glucans extracted from mycelium and fruiting body ofC. unicolorwere also used as potential and specific inducers of mutanase synthesis byTrichoderma harzianum. The highest mutanase activity (0.38 U/mL) was obtained after induction of enzyme by (1→3)-α-d-glucan isolated from the mycelium ofC. unicolor, and this biopolymer has been suggested as a new alternative to streptococcal mutan for the mutanase induction inT. harzianum. (1→3)-α-d-Glucan-induced mutanase showed high hydrolysis potential in reaction with dextranase-pretreated mutan, where maximal degree of saccharification and solubilization of this bacterial homoglucan (83.1% and 78.4%, resp.) was reached in 3 h at 45°C.