Transcriptome Analysis Reveals Candidate Genes Involved in Light-Induced Primordium Differentiation in Pleurotus eryngii.

Pleurotus eryngii, a highly valued edible fungus, is one of the major commercially cultivated mushrooms in China. The development of P. eryngii, especially during the stage of primordium differentiation, is easily affected by light. However, the molecular mechanism underlying the response of primordium differentiation to light remains unknown. In the present study, primordium expression profiles under blue-light stimulation, red-light stimulation, and exposure to darkness were compared using high-throughput sequencing. A total of 16,321 differentially expressed genes (DEGs) were identified from three comparisons. GO enrichment analysis showed that a large number of DEGs were related to light stimulation and amino acid biosynthesis. KEGG analyses demonstrated that the MAPK signaling pathway, oxidative phosphorylation pathway, and RNA transport were most active during primordium differentiation. Furthermore, it was predicted that the blue-light photoreceptor WC-1 and Deoxyribodipyrimidine photolyase PHR play important roles in the primordium differentiation of P. eryngii. Taken together, the results of this study provide a speculative mechanism that light induces primordium differentiation and a foundation for further research on fruiting body development in P. eryngii.

Comparing the removal of polycyclic aromatic hydrocarbons in soil after different bioremediation approaches in relationto the extracellular enzyme activities.

A 120-day experiment was conducted to compare the removal of polycyclic aromatic hydrocarbons (PAHs) from agricultural soil after natural attenuation (NA), phytoremediation (P), mycoremediation (M), and plant-assisted mycoremediation (PAM) approaches in relation to the extracellular enzyme activities in soil. The NA treatment removed the total soil PAH content negligibly. The P treatment using maize (Zea mays) enhanced only the removal of low and medium molecular PAHs. The Pleurotus ostreatus cultivated on 30-50 mm wood chip substrate used in M treatment was the most successful in the removal of majority PAHs. Therefore, significantly (p < 0.05) highest total PAH removal by 541.4 µg/kg dw (dry weight) (36%) from all tested M treatments was observed. When using the same fungal substrate together with maize in PAM treatment, the total PAH removal was not statistically different from the previous M treatment. However, the maize-assisted mycoremediation treatment significantly boosted fungal biomass, microbial and manganese peroxidase activity in soil which strongly correlated with the removal of total PAHs. The higher PAH removal in that PAM treatment could be reflected in the following post-harvest time. Our suggested M and PAM approaches could be promising in situ bioremediation strategies for PAH-contaminated soils.

High temperature induced disruption of the cell wall integrity and structure in Pleurotus ostreatus mycelia.

Fungal cells are surrounded by a tight cell wall to protect them from harmful environmental conditions and to resist lysis. The synthesis and assembly determine the shape, structure, and integrity of the cell wall during the process of mycelial growth and development. High temperature is an important abiotic stress, which affects the synthesis and assembly of cell walls. In the present study, the chitin and ß-1,3-glucan concentrations in the cell wall of Pleurotus ostreatus mycelia were changed after high-temperature treatment. Significantly higher chitin and ß-1,3-glucan concentrations were detected at 36 °C than those incubated at 28 °C. With the increased temperature, many aberrant chitin deposition patches occurred, and the distribution of chitin in the cell wall was uneven. Moreover, high temperature disrupts the cell wall integrity, and P. ostreatus mycelia became hypersensitive to cell wall-perturbing agents at 36 °C. The cell wall structure tended to shrink or distorted after high temperature. The cell walls were observed to be thicker and looser by using transmission electron microscopy. High temperature can decrease the mannose content in the cell wall and increase the relative cell wall porosity. According to infrared absorption spectrum, high temperature broke or decreased the glycosidic linkages. Finally, P. ostreatus mycelial cell wall was easily degraded by lysing enzymes after high-temperature treatment. In other words, the cell wall destruction caused by high temperature may be a breakthrough for P. ostreatus to be easily infected by Trichoderma.

Morphological and molecular characterization of yellow oyster mushroom, Pleurotus citrinopileatus, hybrids obtained by interspecies mating.

Pleurotus citrinopileatus (yellow oyster mushroom) has an attractive shape and yellow colour but the fragile texture complicates packaging, and its strong aroma is unappealing to consumers. This study aimed to improve the characteristics and yield of P. citrinopileatus by interspecies mating between monokaryotic cultures of P. citrinopileatus and P. pulmonarius. Ten monokaryon cultures of the parental lines were crossed in all combinations to obtain hybrids. Eleven compatible mating pairs were obtained and cultivated to observe their sporophore morphology and yield. The selected hybrid, i.e. P1xC9, was beige in colour while hybrid P3xC8 was yellow in colour. Their sporophores had less offensive aroma, improved texture and higher yield. The DNA sequences of these hybrids were found to be in the same clade as the P. citrinopileatus parent with a bootstrap value of 99%. High bootstrap values indicate high genetic homology between hybrids and the P. citrinopileatus parent. The biological efficiencies of these hybrids P1xC9 (70.97%) and P3xC8 (52.14%) were also higher than the P. citrinopileatus parent (35.63%). Interspecies hybrids obtained by this mating technique can lead to better strains of mushrooms for genetic improvement of the Pleurotus species.

Hydrodynamics, Fungal Physiology, and Morphology.

Filamentous cultures, such as fungi and actinomycetes, contribute substantially to the pharmaceutical industry and to enzyme production, with an annual market of about 6 billion dollars. In mechanically stirred reactors, most frequently used in fermentation industry, microbial growth and metabolite productivity depend on complex interactions between hydrodynamics, oxygen transfer, and mycelial morphology. The dissipation of energy through mechanically stirring devices, either flasks or tanks, impacts both microbial growth through shearing forces on the cells and the transfer of mass and energy, improving the contact between phases (i.e., air bubbles and microorganisms) but also causing damage to the cells at high energy dissipation rates. Mechanical-induced signaling in the cells triggers the molecular responses to shear stress; however, the complete mechanism is not known. Volumetric power input and, more importantly, the energy dissipation/circulation function are the main parameters determining mycelial size, a phenomenon that can be explained by the interaction of mycelial aggregates and Kolmogorov eddies. The use of microparticles in fungal cultures is also a strategy to increase process productivity and reproducibility by controlling fungal morphology. In order to rigorously study the effects of hydrodynamics on the physiology of fungal microorganisms, it is necessary to rule out the possible associated effects of dissolved oxygen, something which has been reported scarcely. At the other hand, the processes of phase dispersion (including the suspended solid that is the filamentous biomass) are crucial in order to get an integral knowledge about biological and physicochemical interactions within the bioreactor. Digital image analysis is a powerful tool for getting relevant information in order to establish the mechanisms of mass transfer as well as to evaluate the viability of the mycelia. This review focuses on (a) the main characteristics of the two most common morphologies exhibited by filamentous microorganisms; (b) how hydrodynamic conditions affect morphology and physiology in filamentous cultures; and (c) techniques using digital image analysis to characterize the viability of filamentous microorganisms and mass transfer in multiphase dispersions. Representative case studies of fungi (Trichoderma harzianum and Pleurotus ostreatus) exhibiting different typical morphologies (disperse mycelia and pellets) are discussed.

Production and molecular characterization of somatic hybrids between Pleurotus florida and Lentinula edodes.

Nine inter-generic somatic hybrids named as pfle were produced through PEG-mediated protoplast fusion between Pleurotus florida and Lentinula edodes using double selection method. Hybridity of the newly developed strains was established on the basis of colony morphology, mycelial growth, hyphal traits, fruit-body productivity and inter single sequence repeat (ISSR) marker profiling. Hybrid population was assessed with different phenotypic variables by one-way analysis of variance. Principal component matrices were analyzed for the six phenotypic variables in scatter plot showing maximum positive correlation between each variable for all strains examined. Six ISSR primers generated 66 reproducible fragments with 98.48 % polymorphism. The dendrogram thus created based on unweighted pair-group method with mathematic averages method of clustering and Euclidean distance which exhibited three major groups between the parents and pfle hybrids. Though P. florida parent remained in one group but it showed different degrees of genetic distance with all the hybrid lines belonging to the other two groups while L. edodes was most distantly related to all the hybrid lines. L. edodes specific sequence-rich ISSR amplicon was recorded in all the hybrid lines and in L. edodes but not in P. florida. All the fruit body generating pfle hybrid lines could produce basidiocarp on paddy straw in sub-tropical climate and showed phenotypic resemblance to the P. florida parent.

Extracellular ligninolytic enzymes production by Pleurotus eryngii on agroindustrial wastes.

Pleurotus eryngii (DC.) Gillet (MCC58) was investigated for its ligninolytic ability to produce laccase (Lac), manganese peroxidase (MnP), aryl alcohol oxidase (AAO), and lignin peroxidase (LiP) enzymes through solid-state fermentation using apricot and pomegranate agroindustrial wastes. The reducing sugar, protein, lignin, and cellulose levels in these were studied. Also, the production of these ligninolytic enzymes was researched over the growth of the microorganism throughout 20 days, and the reducing sugar, protein, and nitrogen levels were recorded during the stationary cultivation at 28 ± 0.5°C. The highest Lac activity was obtained as 1618.5 ± 25 U/L on day 12 of cultivation using apricot. The highest MnP activity was attained as 570.82 ± 15 U/L on day 17 in pomegranate culture and about the same as apricot culture. There were low LiP activities in both cultures. The maximum LiP value detected was 16.13 ± 0.8 U/L in apricot cultures. In addition, AAO activities in both cultures showed similar trends up to day 17 of cultivation, with the highest AAO activity determined as 105.99 ± 6.3 U/L on day 10 in apricot cultures. Decolorization of the azo dye methyl orange was also achieved with produced ligninolytic enzymes by P. eryngii using apricot and pomegranate wastes.

[Relationship between mycelium morphology and laccase production of Pleurotus ferulae in submerged cultivation].

In this study, the relationship between mycelium morphology and laccase production was studied. The results indicated that the morphology of P. ferulae pellets was changed when glass beads were added. Laccase production showed higher with spherical mycelium than with filamentous or flocculent mycelium. In addition, the spherical mycelium with a diameter of 0.2-0.4 mm highly affected laccase production. Effect of the composition of culture medium on pellets was investigated and results indicated that various concentrations of glucose, corn meal and wheat bran were important to the formation of pellets in diameter of 0.2-0.4 mm. Besides nutrients, the addition of non-nutritional substrates influenced the distribution of P. ferulae pellets. However, the production of laccase was not promoted by non-nutritional substrates.

Enhanced green fluorescent protein expression in Pleurotus ostreatus for in vivo analysis of fungal laccase promoters.

The laccase family of Pleurotus ostreatus has been widely characterized, and studies of the genes coding for laccase isoenzymes in P. ostreatus have so far led to the identification of four different genes and the corresponding cDNAs, poxc, pox1, poxa1b and poxa3. Analyses of P. ostreatus laccase promoters poxc, pox1, poxa1b and poxa3 have allowed identification of several putative response elements, and sequences of metal-responsive elements involved in the formation of complexes with fungal proteins have been identified in poxc and poxa1b promoters. In this work, development of a system for in vivo analysis of P. ostreatus laccase promoter poxc by enhanced green fluorescent protein expression is performed, based on a poly ethylene glycol-mediated procedure for fungal transformation. A quantitative measurement of fluorescence expressed in P. ostreatus transformants is hereby reported for the first time for this fungus.

Optimization of extracellular glucan production from Pleurotus eryngii and its impact on angiogenesis.

Pleurotus eryngii, an edible mushroom with therapeutic potential was optimized using response surface methodology of four-factor Box-Behnken design for maximum mycelial biomass and extracellular glucan (EPS) production. The model predicts to gain a maximal mycelial biomass and extracellular polysaccharide at 39.4 g/l; 36.04 g/l of glucose, 8.27 g/l; 7.51 g/l of yeast extract, pH 6.99; 7.07 and temperature 26.2°C; 25.84°C, respectively. The validation experiments showed that the model was significant and in close agreement with the model prediction. The evaluation of extracellular polysaccharide on angiogenesis by ex vivo CAM assay showed that there was significant inhibition in neo-vascularization.

Differential proteomic analysis of temperature-induced autolysis in mycelium of Pleurotus tuber-regium.

Autolysis is an important physiological process found in fungal cultivation. However, there is hitherto no report on the autolysis of Pleurotus tuber-regium. We have investigated the enzymes secreted by temperature-induced (40°C as treatment versus 10°C as control) autolysis of the mycelium of P. tuber-regium grown in submerged cultivation. A comparison between the intracellular proteins (inside the mycelium) and the extracellular proteins (in the culture medium) of the treatment and control by proteomic analysis involving 2D PAGE and MALDI-TOF-MS was made. Twenty-two up-regulated protein spots were detected and eight proteins were identified. They included proteasome which participates in the ubiquitin-proteasome pathway; ß-1,3-glucanosyltransferase and tubulin which are involved in the renewal and repair of cell wall; protease and endoglucanase which promote the natural degradation of cell wall and cytoplasm; 14-3-3 protein which takes part in cell signal transduction; and two putative proteins presumably relate to the autolysis process. These identified proteins suggest partially the metabolic processes of the autolysis in the P. tuber-regium mycelium.

Extraction and antioxidant activities of intracellular polysaccharide from Pleurotus sp. mycelium.

The extraction conditions of intracellular polysaccharide (IPS) from Pleurotus sp. mycelium in submerged culture were investigated. Four parameters affecting the IPS extraction, ultrasonic treatment time, extraction temperature, extraction time and ethanol concentration, were determined by single factor tests and then optimized by orthogonal experiments. Under the optimized conditions, the extraction rates of IPS of Pleurotus nebrodensis SJ-02, Pleurotus eryngii SI-01 and Pleurotus corncopiae SS-01 were 7.1+/-0.4%, 7.5+/-0.3%, and 8.2+/-0.5%, respectively. The in vitro hydroxyl radical inhibition percentages of IPS of three mushrooms were 32.2+/-2.8%, 16.1+/-1.5%, and 38.7+/-3.1%, respectively. The scavenging effects of IPS on superoxide anion radical and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical were 19.1+/-1.5%, 16.3+/-1.3%, 20.3+/-1.8%, 17.9+/-1.6%, 16.8+/-1.4%, and 20.5+/-1.7%, respectively. The results provide a reference for large-scale production of IPS by Pleurotus sp. in industrial fermentation.

[Comparisons of different methods for virus-elimination of edible fungi].

Four dsRNA bands were extracted from Pleurotus ostreatus TD300 by the dsRNA isolation technique with sizes of 8.2 kb, 2.5 kb, 2.1 kb, and 1.1 kb, respectively. Four virus-eliminated methods, i. e. hyphal tips cut (HTC), protoplast regeneration (PR), single spore hybridization (SSH), and frozen and lyophilized (FL), were applied to prepare virus-eliminated strains, and one virus-eliminated strain was selected for each virus-elimination method. The virus-eliminated strains were named as HTC8, PR15, FL01, and SSH11, respectively. There were low concentration of 8.2 kb dsRNA remained in HTC8, as well as low concentration of 8.2 kb and 2.5 kb dsRNA remained in FL01. However, no dsRNA remained in PR15 and SSH11. The hyphal growth rate and laccase activity of the virus-eliminated strains increased, especially HTC8 and PR15, whose hyphal growth rate was higher by 22.73% and 18.18%, and laccase activities higher by 145.83% and 134.38% than that of the original strain, respectively. The conclusion is that hyphal tips cut and protoplast regeneration are suitable to prepare virus-eliminated strains of edible fungi.

Characterization and antineoplasic effect of extracts obtained from Pleurotus sajor-caju fruiting bodies.

Fungi of the Pleurotus genus present a great industrial interest due to their possibility of producing pharmacological compounds, pigments, aromas, organic acids, polysaccharides, enzymes, vitamins, amino acids, etc. Among the therapeutic products, we can highlight those with antineoplasic activity, attributed to the fungi cell wall components. Based on this, the objective of this work was to study the antineoplasic capacity of the polysaccharidic fractions obtained from Pleurotus sajor-caju fruiting bodies. Female Swiss mice were inoculated with the Ehrlich ascitic tumor (5 x 10(6) cells/animal) in ascitic form. The polysaccharidic fractions were administered intraperitoneally, during a 6-day period. Fractions FI and FII presented a lower volume of ascitic liquid (3.1 and 1.8 mL, respectively) and a higher reduction in the number of neoplasic cells present in the ascitic liquid (86.2% and 85%, respectively), when compared to the positive control (group inoculated with the tumor but without treatment). These fractions were characterized in terms of monosaccharide composition. Glucose was the major component detected, followed by galactose and mannose. The anomeric carbon configuration of the beta-glucan was confirmed by the (13)C NMR (delta 103.7). Substituted and free C3 and C6 were also detected. Protein bands were confirmed through infrared analysis.

In situ recovery of the aroma compound perillene from stirred-tank cultured Pleurotus ostreatus using gas stripping and adsorption on polystyrene.

Supplementation of the key metabolite, alpha-(Z)-acaridiol, to stirred-tank cultured Pleurotus ostreatus was used to demonstrate that integrated in situ product recovery resulted in high conversion rates and quantitative separation of the target product perillene from the nutrient medium. The conversion of beta-myrcene by P. ostreatus was scaled-up from shake-flasks into a controlled, stirred tank bioreactor equipped with gas stripping and adsorption on a polystyrene fixed bed. The formation of the attractive flavour compound perillene was measured daily using standard controlled capillary gas chromatography. The formation of alpha-(Z)-acaridiol was the metabolic bottleneck of the conversion of beta-myrcene to perillene. Efficient in situ recovery of the volatile product enabled quantitative separation of the pure flavour compound. Appropriated bioprocessing, i.e. in situ separation of product, steadily shifted the metabolic equilibria and thus accomplished high conversion rate and pure product.

[Micromorphological features of Pleurotus pulmonarius (Fr.) Quel. and P. ostreatus (Jacq.) P. Kumm. strains in pure and binary culture with yeasts].

Micromorphology of oyster mushrooms Pleurotus ostreatus (Jacq.) Quel. and P. pulmonarius (Fr.) Quel. was studied in pure and binary culture with yeasts (Cryptococcus laurentii 1629, Rhodotorula minuta 2790, Sporidiobolus salmonicolor (see text for symbol) 31A-11, Candida krusie 3452, Pichia holstii 3438). The cultures were cultivated on malt-agar and water agar. Various mycelial structures were described: strands, rings, thin searching mycelium, clamps, crystals, head-like offshoots, mycelial fragments, chlamydospores, and coralloid hyphae. Vegetative mycelia interact in different ways (forming anastomoses, strands, system of thin anucleate hyphae) within the same culture. Head-like offshoots of mycelial cells, previously regarded as spores of asexual reproduction, appeared to lack nuclei and to be filled with polyphosphates. Coralloid hyphae, which induce yeast cell lysis after direct contact, were detected only in binary culture with yeasts under condition of nitrogen deficit. The same way of feeding is typical for carnivorous mushrooms.

Microscopic observations of the early development of Pleurotus pulmonarius fruit bodies.

From observations made by light microscopy, transmission electron microscopy, environmental-scanning and cryoscanning electron microscopy we conclude that the expansion of the young fruit body of Pleurotus pulmonarius involves considerable vacuolation of hyphae but no marked inflation of cell dimensions. There is evidence for an extensive extracellular matrix (ECM), the components of which must be under the control of the hyphae which the ECM surrounds. However the ECM in these fruit bodies is a dilute material. It is easily lost during specimen preparation and is evident only when certain techniques are used to preserve the fluid surface of the hyphae. Observations of the hyphal and fruit body structures with a range of conventional microscopic techniques are crucial to complement the information obtained through physiological and molecular studies for understanding the cellular changes that occur during mushroom development.

Interaction between gfp-tagged Pseudomonas tolaasii P12 and Pleurotus eryngii.

Pseudomonas sp., (formerly reported as strain P12) which produces brown blotch disease symptoms on Pleurotus eryngii, has been identified as P. tolaasii based on its biochemical, physiological properties and 16S rDNA sequence analysis. This pathogen is able to infect basidiocarps when surface-inoculated on mushroom casing soil. However, infected basidiocarps develop the brown blotch disease symptoms when the pathogen concentration in the fruiting body tissues is higher than 10(4) cfu/g d.w. Using gfp-tagged cells and confocal laser scanning microscopy, it was possible to show that the pathogen has the ability to tightly attach to the hyphae of Pleurotus eryngii.