Fruiting Body Induction Research Articles

Effect of bacterial volatiles on the mycelial growth of mushrooms

Bacteria play an important role in the life cycle of fungi by influencing positively or negatively morphological features, mycelial growth and/or fruiting body induction. However, little is known about the underlying mechanisms and their species-dependence, especially among fungi of the phylum Basidiomycota. Hence, we analyzed the effects of seven bacterial isolates, that were previously obtained from Pleurotus ostreatus HK35, on the mycelial growth of P. ostreatus HK35, Pleurotus eryngii DSMZ 8264, Pleurotus sapidus DSMZ 8266, Pleurotus citrinopileatus DSMZ 5341, Cyclocybe aegerita AAE-3, Lentinula edodes CBS 389.89 and Kuehneromyces mutabilis DSMZ 1013 during eight days. Notably, the bacterial isolates only showed significant mycelial growth-promoting effects when co-cultivated on Petri dishes with Pleurotus species, except for P. citrinopileatus. In particular, Paenibacillus peoriae strain M48F induced remarkably the mycelial growth in P. ostreatus (∼47 %), P. eryngii (∼32 %) and P. sapidus (∼27 %) during the early cultivation stages, but with ongoing cultivation this strain inhibited the growth of all fungi. To investigate the impact of bacterial volatile organic compounds (VOCs) on the mycelial growth, P. ostreatus and P. eryngii were co-cultivated with the bacteria on bi-plates. No growth inhibition on bi-plates was observed while bacterial isolates and mycelia were separated by a physical barrier, assuring that late mycelial growth inhibition was not caused by bacterial volatile compounds. VOCs from strain M48F induced the strongest growth of P. ostreatus (∼50 %) and P. eryngii (∼20 %) mycelia compared to controls. Furthermore, we analyzed the VOCs of strain M48F alone and in combination with P. ostreatus, P. eryngii, P. sapidus and L. edodes using bi-plates and SPME-GC-MS. Strain M48F triggered the formation of β-bisabolene when co-cultivated with P. ostreatus or P. eryngii, which may indicate a fungal defense reaction. Additionally, 2,5-diisopropylpyrazine dominated the volatilome of strain M48F on all eight sampling days. In samples of strain M48F, alone and co-cultivated with L. edodes, the amount of 2,5-diisopropylpyrazine remained quite constant. In contrast, the quantity of this substance declined substantially in co-cultures with P. ostreatus. Interestingly, 2,5-diisopropylpyrazine enhanced P. ostreatus mycelial growth significantly although the growth-promoting effect was not as pronounced as during co-cultivation with strain M48F. Our results show that the mycelial growth-promoting effects of bacteria are remarkably species-dependent, and that bacterial VOCs such as 2,5-diisopropylpyrazine can enhance mycelial growth.

Effect of Spirulina biomass residue on yield and cordycepin and adenosine production of Cordyceps militaris culture

Spirulina biomass residues (SBR) are left behind after pigments extraction. Residual essential nutrients include protein, carbohydrate and lipid. This research focused investigated SBR from phycocyanin extraction as a protein source for Cordyceps militaris culture to replace the use of commercial Eri silk worms and produce better amounts of some valuable nutrients, as well as improve consumer safety. C. militaris samples were cultured on fruiting body induction medium (FIM) using dried Spirulina and dried SBR at 0, 1, 2.5, 5, 10% w/v compared with the FIM with silkworm larvae in the dark at 25 ± 2 °C for 2 weeks, then cultured under illumination (50 μmol/m−2/s−1) for 16 h at 20 ± 2 °C for 10 weeks. The results showed that C. militaris cultured in the FIM supplement with 1% dried SBR produced the highest amount of cordycepin (32.87 mg/g-DW), which was higher than in other studies and in silk worm product.

A Bcl-2 Associated Athanogene (bagA) Modulates Sexual Development and Secondary Metabolism in the Filamentous Fungus Aspergillus nidulans.

The Bcl-2 associated athanogene (Bag) family is a multifunctional group of proteins distinguished by a conserved region known as the Bag domain (BD). Herein, we discuss the discovery and characterization of a Bag protein in the model genetic fungus Aspergillus nidulans, we designated BagA. BagA shares striking similarities in 3D structure, domain organization, amino acid properties, and Hsp70 binding surfaces to animal and plant Bags. While Hsp70 binding is a common feature of Bag proteins, our experimental evidence shows that BagA does not cooperate with A. nidulans Hsp70s, suggesting this association may not be a universal feature of Bag proteins. Gene expression of bagA was strongly induced during sexual development suggesting a role in developmental processes. Accordingly, the deletion of bagA (ΔbagA) negatively impacted sexual development, while its overexpression resulted in constitutive induction of sexual fruiting bodies and spores. Asexual and sexual development was linked to secondary metabolism in A. nidulans. Our data show that the deletion of bagA also provoked an altered secondary metabolite (SM) profile in both sexual and vegetative growth phases. Indeed, LC-MS analysis showed a significant enrichment of SMs in ΔbagA, including novel metabolites not produced by wild type strain. Enrichment of SMs in ΔbagA strain is particularly intriguing and suggest that altering cellular homeostasis can be used as a provocative strategy to activate cryptic metabolites and uncover novel bioactive compounds. Overall, our results indicate that Bag proteins in filamentous fungi share developmental regulatory roles with their animal and plant counterparts. We also show a potentially unique role for BagA in modulating secondary metabolism in A. nidulans. To our knowledge, this study provides a first insight into Bag function in filamentous fungi.

Influencia de la luz azul sobre la productividad del cultivo sólido de Ganoderma lucidum

Ganoderma lucidum es un hongo macromiceto reconocido por sus propiedades medicinales y su contenido de compuestos bioactivos que incluyen polisacáridos, triterpenoides, proteínas inmunomoduladoras, entre otros, lo que ha generado un incremento notable en su producción. La mayoría de especies de hongos responden y se adaptan a diversas señales ambientales incluida la luz, que favorece su productividad, tanto en calidad como en cantidad al estar estrechamente relacionada con la formación de cuerpos fructíferos. Por tal razón, el objetivo de este estudio fue evaluar la eficiencia biológica (EB) y la tasa de producción (TP) como parámetros de productividad del cultivo sólido de Ganoderma lucidum bajo irradiación de los sustratos con luz emitida por diodos azules (LED) con dos periodos de foto-estímulo de 12 y 24 h durante todas las fases de cultivo para inducir el crecimiento micelial y la formación de los cuerpos fructíferos. Se aplicaron parámetros convencionales para el crecimiento y desarrollo del hongo en las etapas de producción. Para la formulación de los sustratos, se emplearon residuos agroindustriales y materiales lignocelulósicos. El diámetro de los cuerpos fructíferos sometidos a tratamientos con luz azul fue mayor que los exhibidos a luz blanca fluorescente (Testigo). Los resultados muestran que el cultivo de Ganoderma lucidum con exposición a la luz azul es útil para la inducción de cuerpos fructíferos de alta calidad, logrando una disminución del periodo de fermentación en 16 días para el foto-estímulo de 24 h con EB de 28,04% y TP de 0,64.

Characterisation of the large-scale production process of oyster mushroom (Pleurotus ostreatus) with the analysis of succession and spatial heterogeneity of lignocellulolytic enzyme activities

Oyster mushroom (Pleurotus ostreatus) lignocellulolytic enzyme activity pattern and variation was investigated in a large-scale facility from spawning until the end of the second flush. In the first cultivation cycle laccase production reached its peak during vegetative growth stage, while manganese-peroxidase showed the highest activity during fruiting body induction. Cellulose and hemicellulose degrading enzymes had maximal activity at the beginning of flush and harvest stage. The enzyme activities showed similar tendencies among five different mushroom substrate blocks representing a production house. The spatial variability analysis of enzyme activities pointed out the within substrate block heterogeneity as the main source if variation. This result was confirmed by Combined Cluster and Discriminant Analysis (CCDA) method showing minimal among block heterogeneity considering the whole investigation period; furthermore in the first cultivation cycle all blocks were grouped into one cluster.

LED lights increase bioactive substances at low energy costs in culturing fruiting bodies of Cordyceps militaris

Cordyceps militaris is widely cultured to produce fruiting bodies for medicinal and health food use. Illumination is an important factor for the growth, quality and production costs. The effects of different light-emitting diode (LED) light combinations on primordia induction, the growth and bioactive substances of fruiting bodies were investigated. The results showed that the monochromatic red or far-red LED failed to induce primordia. Blue light was a must for primordia induction and a red:blue 2:1 with 10% far-red induced the greatest frequency of primordia. Dry weights of fruiting bodies were lower under red and far-red LED treatments. Cordycepin contents in fruiting bodies were significantly higher in red and white (red:blue:far-red 6:3:1) LED treatments, with the increases up to 62% over the fluorescent control. Carotenoids content was significant lower in the red or far-red LED treatment than in other treatments. The results demonstrated the functions of different wavelength lights on the growth and biosynthesis of bioactive substances and that using the low heat release and energy consumption LED lights to replace the traditional fluorescent lights can improve the product quality and increase the contents of bioactive substances. A red to blue ratio of 2:1 with 10% far-red is the optimal light quality for culturing fruiting bodies of C. militaris.

Effect of Electrical Stimulation on Fruit Body Formation in Cultivating Mushrooms.

The effect of high-voltage electrical stimulation on fruit body formation in cultivating mushrooms was evaluated using a compact pulsed power generator designed and based on an inductive energy storage system. An output voltage from 50 to 130 kV with a 100 ns pulse width was used as the electrical stimulation to determine the optimum amplitude. The pulsed high voltage was applied to a sawdust-based substrate of Lyophyllum decastes and natural logs hosting Lentinula edodes, Pholiota nameko, and Naematoloma sublateritium. The experimental results showed that the fruit body formation of mushrooms increased 1.3–2.0 times in terms of the total weight. The accumulated yield of Lentinula edodes for four cultivation seasons was improved from 160 to 320 g by applying voltages of 50 or 100 kV. However, the yield was decreased from 320 to 240 g upon increasing the applied voltage from 100 to 130 kV. The yield of the other types of mushrooms showed tendencies similar to those of Lentinula edodes when voltage was applied. An optimal voltage was confirmed for efficient fruit body induction. The hypha activity was evaluated by the amount of hydrophobin release, which was mainly observed before the fruit body formation. The hydrophobin release decreased for three hours after stimulation. However, the hydrophobin release from the vegetative hyphae increased 2.3 times one day after the stimulation.

Biological characteristics of teleomorph and optimized in vitro fruiting conditions of the Hoelen medicinal mushroom, Wolfiporia extensa (Higher Basidiomycetes).

Wolfiporia extensa is a basidiomycetous brown rot fungus and is of well-known medicinal import in China, Japan, and other Asiatic countries. Fruiting body induction is of major relevance for basic biological research and for their use in industrial applications. Based on the evaluation of the effects of temperature, time in the dark before induction and culture, and wounding treatment on fruiting, this report describes the most efficient protocol for inducing fruiting of W. extensa growing on agar plates. Furthermore, several biological characteristics of teleomorph, such as the locations of hymenium, the configuration of basidiospores and primary mycelia, and events involved in basidiosporogenesis in W. extensa, were analyzed for the first time using fluorescence microscopy. The results showed that the hymenium born on both sides of the wall of the honeycomb-like structure on the surface of fruiting bodies and the hymenophoral trama situated in the middle. Each basidia has 4 binuclear basidiospores, and the primary mycelia are multinucleate without clamp connections. These results broaden our knowledge about this brown rot fungus and promote further studies of the sexual reproduction, fruiting body development, and advancement of breeding program, new topics related to the contents of pharmacologically active substances in W. extensa fruiting bodies.

Protein expression during <i>Flammulina velutipes</i> fruiting body formation

Formation of the Flammulina velutipes fruiting body can be induced by lowering the ambient temperature (first treatment) in complete darkness. Fruiting bodies formed under these conditions elongate without pileus formation (pinhead fruiting body), suggesting that they cannot mature in complete darkness. However, after light treatment of the pinhead fruiting body (second treatment), a pileus develops immediately, and the stipe also thickens and becomes increasingly pigmented. The apical region swells as a result of cell division starting 2 days after light treatment, the pileus–stipe junction fracture and hymenium primordia form on day 4, and gills appear at day 6. Pf1 and Pf3 are specifically expressed after exposure to low temperature without light. The cell wall-associated protein [pileus-specific hydrophobin-like protein (PSH)] is specifically induced in the pileus, but not in the stipe, following the second light treatment to the pinhead fruiting body. These results suggest that Pf1 and Pf3 would be involved in fruiting body induction and that PSH would be involved in pileus formation. These phenomena will aid further histological and molecular biological investigations into the mechanisms behind fruiting body development in F. velutipes.

An Active Compound for Fruiting Body Induction

An Active Compound for Fruiting Body Induction

An Active Compound for Fruiting Body Induction

An Active Compound for Fruiting Body Induction

Biotechnology for in vitro growing of edible and medicinal mushrooms on wood wastes

The aim of this work was focused on finding out the best way to convert the wood wastes into useful food supplements, such as mushroom fruit bodies, by using them as growing sources for the edible and medicinal mushrooms. According to this purpose, three fungal species from Basidiomycetes, namely Ganoderma lucidum (Curt.:Fr.) P. Karst, Lentinus edodes (Berkeley) Pegler and Pleurotus ostreatus (Jacquin ex Fries) Kummer were tested to determine their biological potential to grow on substrates made of wood wastes (sawdusts as well as shavings) which could be used in this way as main ingredients for preparation of natural culture composts. The experiments were achieved by in vitro growing of all these fungal species in special rooms, where the main culture parameters were kept at optimal levels in order to get the highest production of mushroom fruit bodies. The effects of culture compost composition (carbon, nitrogen and mineral sources) as well as other physical and chemical factors (such as: temperature, inoculum amount, pH level and incubation time, etc.) on mycelial net formation and especially on fruit body induction, were investigated. From all these fungal species tested in our experiments, P. ostreatus was registered as the fastest mushroom culture, then L. edodes and finally, G. lucidum as the longest mushroom culture. During the experiments, different logs of the same species were used as control samples for each culture compost variants. Applying such biotechnology, the environmental problems generated by the plant wastes accumulation in wood industry could be solved only by using biological means for their valorising, simultaneously with food supplements producing having high nutritive values as well as healing effects by increasing the consumers` health.

Effect of Casing Layer on Growth Promotion of the Edible Mushroom Pleurotus ostreatus

Various bacteria were isolated from the casing layer soil of the culture bed of P. ostreatus and their role in fruiting body induction of the edible mushroom, P. ostreatus, was investigated. Analysis of the bacterial community isolated from the casing layer soil revealed that the composition of genera and number of cultivable bacteria were different for each sterilizing treatment. Bordetella was predominant in the bulk soil whereas Flavobacterium was predominant after sterilization of the casing layer soil. Fluorescent Pseudomonas was predominant in the non-sterilized casing layer soil. Total number of the bacterial genera in the casing layer soil was higher than that in the bulk soil. In particular, an increase in the fluorescent Pseudomonas population was observed in the non-sterilized casing layer accompanied by induction of fruiting body and enhanced mushroom production yield. The results suggested that specific bacterial populations in the casing layer play an important role in the formation of primodia and the development of basidiome in P. ostreatus.

Structure-Activity Relationships of Triterpenoid Saponins on Fruiting Body Induction inPleurotus ostreatus

To elucidate the structure-activity relationships of saponin on fruiting body induction in Pleurotus ostreatus, monodesmosidic saponins comprising a triterpenoid and glucose residues were synthesized. Betulin, a triterpenoid present in Birch bark, was used as the aglycon. The fruiting body induction activity increased with the number of glucose residues, from one to four. The most active betulinic saponin was betulin-3-yl beta-D-(4-O-beta-D-maltotriosyl)-glucoside.

The Growing Effect of Vineyard and Winery Wastes on the Production of Mycelia and Fruit Bodies of Edible and Medicinal Fungi

It is well known that huge amounts of waste and residue are produced annually during agroindustrial activities, and their utilization could allow self-sustainable processes and products, having a beneficial effect on soil as efficient environmental protection. Many of the lignocellulosic wastes cause serious environmental pollution effects if they are allowed to accumulate in the vineyards, or much worse, to be burned on the soil. At the same time, these wastes could provide an environmentally efficient way to solve the problem by the application of micro-organisms and processes in vine waste treatment and management.The main aim of this work was to find the best way of recycling vine wastes by using them as a growing source for edible and medicinal mushrooms, in order to extend the food chain in vineyard ecosystems. According to this purpose, two fungal species from the Basidiomycetes group—Lentinus edodes (Shiitake) and Pleurotus ostreatus (Oyster Mushroom)—have been used to determine the growing effect of lignocellulosic vineyard wastes used as culture composts as well as winery wastes on the production of mycelia and fruit bodies that could be processed and marketed as useful products, such as food and drugs.The experiments of this research work were achieved by growing all these fungal species in special culture rooms, where all the culture parameters were kept at optimal levels in order to get the highest production of fruit bodies. During the experiments, the effects of culture compost composition (carbon, nitrogen, and mineral sources) as well as other physical and chemical factors (such as temperature, inoculum size, CO2 and O2 concentration, air humidity, watering, light intensity, incubation time) on mycelial net formation and, especially, on fruit body induction were investigated.The registered data revealed that lignocellulosic vine wastes can be used as substrates for mushroom growing only after some mechanical pretreatments to break down the whole lignocellulose structure in order to be more susceptible to fungal enzyme action. All these pretreated lignocellulosic wastes were disinfected by steam sterilization at 120 deg;C for 60 minutes. The final composition of culture composts was improved by adding grain seeds (wheat, rye, rice), CaCO3, and NH4H2PO4, to each kind of culture medium composition, depending on the fungal species used to be grown.All the culture composts for mushroom growing were inoculated using liquid inoculum, aged 5−7 days, and the volume size ranging between 5% and 7% (v/w).The optimal temperatures for incubation and mycelia growth were maintained between 23 °C and 25 °C. The whole period of mushroom growing from inoculation to fruit body formation lasted between 15 and 35 days, depending on each fungal species used in experiments. From all these fungal species tested in our experiments, Pleurotus ostreatus was registered as the fastest mushroom culture (15−20 days), in comparison with the fungal strains of Lentinus edodes (25−30 days).As control samples for each variant of culture composts used for experimental growth of all these fungal species, we used wood chips of the oak that were kept in water 3 days before the experiments and were then steam sterilized for disinfection. Because of their high content of carbohydrates and nitrogen, the variants of culture composts supplemented with wheat grains at a ratio of 1:10 and rice grains at the ratio 1:5 as well as a water content of 60% were optimal for the fruit body production of Pleurotus ostreatus and, respectively, Lentinus edodes. In addition, some strains of this fungal species were grown in our experiments on culture substrates made of winery wastes and rye grains at a ratio of 1:7 and a water content of 50%. A higher ratio of rye grains might lead to an increase of total dry weight of the fruit body, but also could induce the formation of smaller fruit bodies than those of the control samples.The results achieved by using propylene-bag cultures revealed that the composition of culture substrate had significant effects on fruit body production as well as on the characteristic shape of the fruit bodies. The final fruit body production of these fungal species used in experiments was registered between 7 and 10 kg, relative to 100 kg of compost.

Growth promotion of the edible fungus Pleurotus ostreatus by fluorescent pseudomonads

Bacteria were isolated from the mycelial surface of Pleurotus ostreatus and their role in fruiting body induction (fructification) of the edible mushroom P. ostreatus was investigated. Analysis of the bacterial community that colonized the mycelium showed that the species composition and numbers of culturable bacteria differed according to the developmental stage of P. ostreatus. In particular, the population size of fluorescent pseudomonads increased during fruiting body induction. An experiment showed that inoculation of pure cultures of the mycelium with strains of fluorescent Pseudomonas spp. isolated from the mycelial plane of commercially produced mushrooms promoted the formation of primordia and enhanced the development of the basidiome of P. ostreatus. Results of this research strongly suggest that inoculation of the mycelium with specific bacteria may have beneficial applications for mushroom production.

Protein expressions during fruit body induction of Flammulina velutipes under reduced temperature

The influences of temperature on the fruit body induction of Flammulina velutipes were investigated. The fruit body could be induced after reducing of the ambient temperature under complete darkness, but could not be induced after light irradiation without temperature reduction. Fruit bodies formed under complete darkness elongated without pileus formation (pinhead fruit body). Protein expression patterns were investigated by two-dimensional electrophoresis, and 22 protein spots were found to be newly expressed in the mycelia and fruit bodies after reducing of the ambient temperature, but few protein spots were newly expressed in the mycelia after light irradiation without temperature reduction. Four of the spots had identical N-terminal sequences to polypeptides, Pf1, Pf3, Pfd3, and Pf6 which are previously sequenced as polypeptides expressed specifically during fruit body formation of F. velutipes.

Light-induced fruit body formation of an entomogenous fungus Paecilomyces tenuipes

Asexual fruit body formation of an entomogenous fungus, Paecilomyces tenuipes, on an artificial culture medium was investigated. For the fruit body induction, light/dark condition was important. Illumination by white light in a particular critical period preceded by a dark period induced the fruit bodies. The critical period was identified to be around 6–14 d after inoculation at 20°C

The Agaricus bisporus hypAGene Encodes a Hydrophobin and Specifically Accumulates in Peel Tissue of Mushroom Caps during Fruit Body Development

Differential screening of a cDNA library was used to clone genes that are specifically expressed during mushroom development in the basidiomycete Agaricus bisporus. One of the isolated genes encodes a polypeptide of 112 amino acid residues and belongs to the fungal gene family encoding hydrophobins. This gene, hypA, has the characteristic pattern of eight cysteine residues at conserved positions and a hydrophobicity pattern that is very similar to class I hydrophobins. Elucidation of the genomic structure of hypAled to the identification of a second copy, hypC, located downstream of hypA. Although at a much lower level, hypCis like hypAspecifically expressed in fruit bodies. The hypAmRNA level is transiently increased ten days after fruit body induction and expression appears to be associated with rapid expansion of the mushroom caps. In mushroom caps, very high concentrations of hypAmessengers were found in the (outer) peel tissue, where they accumulate to more than 60% of the total mRNA mass. The corresponding protein with a molecular mass of 8 to 9 kDa was purified from this peel tissue and was identified by N-terminal sequencing. Our results suggest that HYPA forms a protective hydrophobic layer instrumental in cap formation.

Induction of fruiting bodies in a macrocyst-forming mutant of the cellular slime mold, Dictyostelium mucomides

Some wild-type strains of Dictyostelium mucoroides exhibit dimorphism in development depending on culture conditions: on agar, fruiting bodies containing stalk and spore cells are formed, whereas under water, a thick-walled structure lacking spore and stalk cells (the macro-cyst) is formed. The mutant, MF-1, was derived from one of these wild-type strains. It forms macrocysts on an agar surfxe as well as under water. It was found that MF-1 could be induced to form fruiting bodies in two ways. First, when an aggregation center from the wild-type strain was grafted to an MF-1 aggregation center. MF-1 cells migrated to the center and formed a large aggregate that gave rise to many slugs that became fruiting bodies. This result, along with the observation that MF-1 aggregates have no tip, suggests that MF-1 normally produces an aggregation center that is unable to organize the aggregate to form a slug. Second, when MF-1 cells were allowed to develop on 1.2 m M ethionine (an analog of methionine), they formed aggregates with tips and developed into fruiting bodies with thick stalks instead of macrocysts. The effect of ethionine was blocked by the presence of 2.4 m M methio-nine. Two other methionine analogs were also tested, i.e., α-methylmethionine and norleucine. When cultured on the former at concentrations ranging from 1.2 to 9.6 m M, MF-1 cells still produced macrocysts; when cultured on norleucine at concentrations ranging from 2.4 to 9.6 m M, MF-1 cells aggregated into large clumps that formed numerous slugs, but these failed to continue development to fruiting bodies. In vertebrates, it is known that a major biochemical effect of ethionine is the inhibition of the methylation of nucleic acids, proteins, and phospholipids. Norleucine and a-methylmethionine inhibit methylation to a lesser extent. Thus, it can be speculated that the biological effects of ethionine on MF-1 cells may result from its interference with methylation reactions, suggesting that macrocyst formation may involve excess methylation as compared with the situation during fruiting-body development.