Changes in phenol oxidases and superoxide dismutase during fruit-body formation of <Emphasis Type="Italic">Pleurotus </Emphasis>on sawdust culture

 Peroxidase and laccase activities increased rapidly up to the formation of primordia and then declined throughout the entire stage of fruiting. In the case of Pleurotus ostreatus, the level of Mn-dependent peroxidase was very low in primordia and fruiting stages but gradually increased with the growth of the fruit-body, whereas no activity was detected in Pleurotus sajor-caju during all growth stages. Superoxide dismutase activity was observed mainly at the fruiting stages. These results show that changes in concentration of lignin-related enzymes are associated with the fruiting process. Received: December 11, 2000 / Accepted: March 28, 2002     

Lignocellulolytic enzymes profile during growth and fruiting of Pleurotus ostreatus on wheat straw and tree leaves

Cultivation of two commercial Pleurotus ostreatus (oyster mushroom) strains was performed in plastic bags. Tree leaves appeared to be an excellent growth substrate for the conversion into fruiting bodies with biological efficiency of 108-118%. The level of enzyme activity was strongly regulated during the life cycle of mushrooms. However, despite the quantitative variations, each strain had a similar pattern of enzyme accumulation in fermentation of both substrates. Laccase and MnP activities were high during substrate colonization and declined rapidly during fruiting body development. On the contrary, in substrate colonization P. ostreatus expressed comparatively low activity of hydrolases. When primordia appeared, the activity of these enzymes sharply increased. Both cellulase and xylanase activity peaked at the mature fruiting body stage. When mushrooms shifted to the vegetative growth, the activity of ligninolytic enzymes again gradually increased, whereas the activity of hydrolases decreased.     

Pleurotus and Agrocybe hemolysins,new proteins hypothetically involved in fungal fruiting

Novel hemolytic proteins, ostreolysin and aegerolysin, were purified from the fruiting bodies of the edible mushrooms Pleurotus ostreatus and Agrocybe aegerita. Both ostreolysin and aegerolysin have a molecular weight of about 16 kDa, have low isoelectric points of 5.0 and 4.85, are thermolabile, and hemolytic to bovine erythrocytes at nanomolar concentrations. Their activity is impaired by micromolar Hg²⁺ but not by membrane lipids and serum low-density lipoproteins (LDL). The sequence of respectively 50 and 10 N-terminal amino acid residues of ostreolysin and aegerolysin has been determined and found to be highly identical with a cDNA-derived amino acid sequence of putative Aa-Pri1 protein from the mushroom A. aegerita, Asp-hemolysin from Aspergillus fumigatus, and two bacterial hemolysin-like proteins expressed during sporulation. We found that ostreolysin is expressed during formation of primordia and fruiting bodies, which is in accord with previous finding that the Aa-Pri1 gene is specifically expressed during fruiting initiation. It is suggestive that the isolated hemolysins play an important role in initial phase of fungal fruiting.     

Beta-adenosine, a bioactive compound in grass chaff stimulating mushroom production

Fructification and yield of the edible mushrooms Pleurotus pulmonarius and Stropharia rugosoannulata are clearly enhanced when wheat straw is supplemented with 30% Lolium perenne grass chaff. The bioactive compound in the methanol extract of grass chaff was identified as beta-adenosine. In vitro biological activity tests showed that 0.012 mg of beta-adenosine per ml of medium stimulated earlier fructification of Pleurotus pulmonarius. Mushroom fruiting trials showed that when 12 mg beta-adenosine was added to 1 kg wet wheat straw, primordia of Pleurotus pulmonarius appeared two days earlier and primordia of Stropharia rugosoannulata appeared 18 days earlier when compared to pure wheat straw substrate. This concentration of beta-adenosine had no impact on the mushroom yield of Pleurotus, but resulted in a 2.2 fold increase in yield for Stropharia. beta-Adenosine at 25 mg per kg wet wheat straw increased the yield of Pleurotus with 52% and the yield of Stropharia with 258%, but this concentration delayed primordial formation in Pleurotus.     

Isolation of high quality and yield of RNA from Agaricus bisporus with a simple, inexpensive and reliable method

A method for isolating high purity and quantity RNA from Agaricus bisporus which is rich in proteins, carbohydrate, fiber and secondary metabolites, is described. RNA was extracted from mycelium, primordia, sporophores at two development stages and two post-harvest storage stages as well as from pileipellis, inner cap, gill and stipe of the mature sporophore. The A(260)/A(230) and A(260)/A(280) ratios of isolated RNA from fruiting bodies were both ~2 and the yield was about 200 μg/g fresh wt (FW). The yield of RNA from mycelium was approx. 100 μg/g FW. High quality RNA was also extracted from fruiting body tissues of Lentinus edodes, Pleurotus ostreatus, Flammulina velutipes and Pleurotus eryngii with yields from 130 to 225 μg/g FW. RNA extracted from all samples was intact, as demonstrated by gel electrophoresis and was suitable for downstream molecular applications, including RT-PCR and qPCR.     

Development of antler-type fruiting bodies of Ganoderma lucidum and determination of its biochemical properties

Following the importance of antler-type fruiting bodies of Ganoderma lucidum, in this study, the impact of main growth parameters such as ventilation and light on the development of antler-type fruiting bodies has been investigated together with the determination of physico-chemical properties of antler fruiting bodies. For this, the primordia bags of G. lucidum were kept under controlled ventilation to adjust the CO₂ produced by the mushrooms owing to its respiration under light and dark conditions. The bioactive compounds such as phenolics, flavonoids, water-soluble polysaccharides and ganoderic acid showed a two-fold increase in the antler-type fruiting bodies as compared to normal kidney-shaped fruiting bodies. It is assumed from this study that the antler type fruiting bodies are developed due to restricted ventilation which causes an increase in the level of CO₂ gas in the air as a result of respiration of mushroom. The shape and colour of antler fruiting bodies again dependent on the light provided in the growth chamber. This study also proves that with the manipulation of light and ventilation antler-type fruiting bodies of G. lucidum could be developed with higher quantity of bioactive compounds and with higher antioxidant potential.     

Heat shock protein 9 mRNA expression increases during fruiting body differentiation in Grifola frondosa and other edible mushrooms

We isolated a homolog of Schizosaccharomyces pombe HSP9 from Grifola frondosa and designated it Gf.HSP9. The Gf.HSP9 gene consisted of four exons and three introns and encoded 84 amino acid residues. We have also identified related HSP9 genes from Pleurotus eryngii (Pe.HSP9), Hypsizygus marmoreus (Hm.HSP9), and Lentinula edodes (Le.HSP9). The predicted tertiary structures of these HSP9s were the same. In addition, the expression pattern of their mRNA increased during fruiting body in a differentiation-dependent manner. These results suggested that HSP9 was associated with the fruiting body differentiation in these four edible mushrooms.     

Yellow Blotch of Pleurotus ostreatus

Yellow blotch disease of the oyster mushroom (Pleurotus ostreatus) was first observed in a commercial mushroom farm in California in 1983. The disease, caused by Pseudomonas agarici, is characterized by primordia, with yellow droplets on their surface, which become stunted, yellow to orange, and deformed as they mature.     

Yield,size, nutritional value,and antioxidant activity of oyster mushrooms grown on perilla stalks

Perilla is an edible medical plant with rapidly increasing acreage in China. In this study, we investigated the potential of perilla stalks (PSs) as an alternative substrate for the cultivation of oyster mushrooms (Pleurotus ostreatus). P. ostreatus was cultivated on cottonseed hulls (CSH) alone or mixed with PSs in different ratios. The production parameters, physical characteristics, nutritional values, and antioxidant activity of mushrooms cultivated on different substrate mixtures were determined. The addition of PSs to CSH significantly improved the growth rate, yield, biological efficiency, and proximate composition and shortened the cultivation cycle. Cultivation on PSs alone increased the amino acid content in P. ostreatus fruiting bodies and the antioxidant activity of mushroom extracts. The PS75 (25% CSH + 75% PS) substrate was deduced to be the most effective substrate on the basis of yield and biological efficiency obtained in a large area where perilla had been planted. The results demonstrate that mixtures of PS with CSHs could be used as novel, practical, and easily accessible alternative substrates for P. ostreatus cultivation.     

Studies on genus Pleurotus. VIII. Interaction between mycelial growth and yield

This project studies the relationship between mycelial growth rate and production of basidiomata of 19 Pleurotus strains. Firstly, monosporic cultures were isolated of five strains from the following species: Pleurotus djamor (3), Pleurotus ostreatus (1) and Pleurotus pulmonarius (1). These were self-crossed in order to obtain 25 infraspecific dikaryons from which their mycelial growth rate was estimated. The parent strains and the 14 fastest growing crosses were cultivated in the pilot plant on barley straw with the following data recorded: days of incubation, primordia initiation, number of harvests, biological efficiency (BE), production rates (PR) and size of the basidiomes. The BE's fluctuated between 16.8 to 75.6% and the PR's between 0.34 to 1.68%. Most of the basidiomata presented a pileus diameter of 5-15 cm. With the exception of one cross with P. djamor, no increase was observed in the productivity and size of the carphophores of the crosses with respect to the parent strains, suggesting that the rapid mycelial growth rate of the strains was not reflected in the development of the fruiting bodies.     

Suppression of Tumor Growth by Pleurotus ferulae Ethanol Extract through Induction of Cell Apoptosis,and Inhibition of Cell Proliferation and Migration

Cancer is the second leading cause of death worldwide. Edible medicinal mushrooms have been used in traditional medicine as regimes for cancer patients. Recently anti-cancer bioactive components from some mushrooms have been isolated and their anti-cancer effects have been tested. Pleurotus ferulae, a typical edible medicinal mushroom in Xinjiang China, has also been used to treat cancer patients in folk medicine. However, little studies have been reported on the anti-cancer components of Pleurotus ferulae. This study aims to extract bioactive components from Pleurotus ferulae and to investigate the anti-cancer effects of the extracts. We used ethanol to extract anti-cancer bioactive components enriched with terpenoids from Pleurotus ferulae. We tested the anti-tumour effects of ethanol extracts on the melanoma cell line B16F10, the human gastric cancer cell line BGC 823 and the immortalized human gastric epithelial mucosa cell line GES-1 in vitro and a murine melanoma model in vivo. Cell toxicity and cell proliferation were measured by MTT assays. Cell cycle progression, apoptosis, caspase 3 activity, mitochondrial membrane potential (MMP), migration and gene expression were studied in vitro. PFEC suppressed tumor cell growth, inhibited cell proliferation, arrested cells at G0/G1 phases and was not toxic to non-cancer cells. PFEC also induced cell apoptosis and necrosis, increased caspase 3 activity, reduced the MMP, prevented cell invasion and changed the expression of genes associated with apoptosis and the cell cycle. PFEC delayed tumor formation and reduced tumor growth in vivo. In conclusion, ethanol extracted components from Pleurotus ferulae exert anti-cancer effects through direct suppression of tumor cell growth and invasion, demonstrating its therapeutic potential in cancer treatment.     

Studies on the Biology of Macrofungi with Reference to their Cultivation

Nutrition, pH, temperature, moisture, light, and aeration as factors influencing mycelial growth, primordia formation, and development of sporophores of 25 species of basidiomycetous macrofungi were studied, of which 18 species produced normal or nearly normal fruit-bodies in the laboratory. It was found that various agricultural by-products and farm wastes, such as sawdust, wood shavings, fallen leaves, soybean hay, bean pods, corn culms, paddy straw, rice glumes, wild grasses, etc. enriched with 20%–25% by weight of rice or wheatbran, made suitable substrata for most of the lignicolous species investigated with the exception of Tremella fuciformis and Lentinus edodes, and that well fermented dungs of domestic animals, such as horse, cow, pig, sheep, rabbit, and poultry, appeared to be satisfactory substitutes for bran as accelerators for fructification. Yields of carpophores were more or less directly proportional to the percentages of accelerators added to the substratum. The vegetative growth of the majority of species studied took place over a wide pH range of 3.0–8.0, with the optimum at 5–6, while the optimal pH for Ganoderma lucidum, Lentinus edodes, and Hericium erinaceus being about 4.0 and the maximum pH limit for H. erinaceus only 5.4. In Auricularia hispida, the pH range for fructification and that for mycelial growth were almost the same, but in Collybia velutipes and Agaricus bitorquis the pH range for sporogenesis was much more restricted, the optimal being 5–6 and 7–8 respectively. Most of the species investigated produced mycelium within a temperature range of 6–36℃, the optimum being about 24–27℃. However, the optimal temperatures for Auricularia auricula, Poria cocos and Volvariella volvacea var. heimii were found to be 30℃ or more. The temperatures required for sporophore and for primordia formation varied from species to species. While the fruit-bodies of Lentinus edodes and Pleurotus sapidus appeared only after "chilling", temperature fluctuations did not exert any stimu- lating effect on primordia differentiation in some other species. Using the temperature requirement for the initiation of primordia as the criterion, the species studied were grouped into the following categories: (1) high-temperature species, such as Ganoderma lucidum, Pleurotus rhodophyllus, Collybia radicata, Volvariella bombycina, and Volvariella volvacea var. heimii, with their optimum and maximum tem- peratures for primordia initiation exceeding 24℃ and 30℃ respectively; (2) intermediate species, such as Auricularia auricula, Tremella fuciformis, Pleurotus citrinopileatus, Pholiota adiposa, Agaricus bitorquis, and Agaricus rubellus, with their maxima not exceeding 28 ℃ and with the optimum lying within 20–24 ℃; and (3) low-temperature species, such as Auricularia hispida, Hericium erinaceus, H. caput-medusae, Pleurotus sapidus, Lentinus edodes, Collybia velutipes, and Agaricus bisporus, with their maxima not exceeding 24 ℃ and with the optimum under 20 ℃. Thus, under natural climatic conditions of a given region, without artificial heating or cooling, it was deemed practicable to arrange, according to the temperature response of the various species, a series of mushroom crops to be grown in rotation the year round. Moisture was found to have considerable influence on vegetative growth as well as reproduction. Most of the lignicolous species grew well on the sawdust medium, which contained 100%–340% by weight of water. Moisture content between 260%–340% was more favourable for fruiting, and several flushes were secured. At 100% moisture, fruiting was considerably delayed and only one flush of poorly developed carpophores was obtained. Aeration also played an important role in basidiocarp development. Stagnant humid air or CO2-laden atmosphere markedly inhibited pileus development, diminishing the size of pilei and causing morphological abnormalities. Adequate supply of fresh air with 80%–90% relative humidity tended to maintain a normal rate of transpiration, thereby guaranteeing a flourishing crop. In some species, such as Ganoderma lucidum, Tremella fuciformis, Pleurotus sapidus, Pleurotus citrinopileatus, Volvariella bombycina, Hericium erinaceus, Auricularia hispida, Armillaria mucida, and Lentinus edodes, light was found to be absolutely necessary. In these species, primordia formation would not commence until exposed to light. Certain other species, such as Auricularia auricula, Pleurotus rhodophyllus, Collybia velutipes, Pholiota adiposa, and Agaricus bitorquis, were able to fructify in total darkness, though their primordia formation was promoted by light. For the normal development and pig- mentation of fruit-bodies, light appeared to be essential. Morphological abnormalities, such as long slender stipes and thin rudimentary pilei occurred in total darkness. Results of experiments indicated that in most of the species studied, light reduced mycelial growth. The inhibitory effect was mainly due to the blue region (380–540 mμ) of the visible spectrum rather than the red region (570–920 mμ). On the contrary, light at the blue end was shown to be most effective and even essential for fructification; the red end being similar to darkness, evoked almost no response. Having analyzed the results obtained, it seems reasonable to assume that the ontogeny of higher Basidiomycetes consists most probably of four developmental stages, viz., (1) the stage of vegetative growth, (2) the stage of primordia initiation, (3) the stage of sporophore development, and (4) the stage of basidiospore formation, each of which has its own physiological characteristics and metabolic speciality.Hence the requirements of the various stages differ considerably.     

Monokariotic fruiting body and clamp cell formation in Mycoleptodonoides aitchisonii (Bunaharitake)

The ability to produce monokaryotic fruiting bodies and clamp cells in culture was examined in monokaryotic strain isolated from several dikaryotic parental strains of the edible mushroom, Mycoleptodonoides aitchisonii (Bunaharitake). We describe a single dikaryotic M. aitchisonii strain, TUFC50005, and 20 monokaryons derived from it, which exhibited a wide spectrum of monokaryotic fruiting types. Most strains formed primordia, or young fruiting body-like structures, but only one of the monokaryons, strain TUFC50005-4, formed a fruiting body, even though it had only one nucleus and produced only two spores after meiosis. We demonstrated that dikariotization was not required for clamp cell formation, fruiting body formation, or meiosis, in this mushroom.     

Effect of light and reductones on differentiation of <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

Vegetative mycelia of Pleurotus ostreatus were differentiated into primordia and subsequently into fruit bodies in synthetic sucrose-asparagine medium when exposed to light at low temperature. During photo-morphogenesis, l-ascorbic acid-like substances called reductones were produced. l-Ascorbic acid, d-eryth-roascorbic acid, 5-O-(α-d-glucopyranosyl)-d-erythroascorbic acid, 5-O-(α-d-xylopyranosyl)-d-erythroascorbic acid, 5-methyl-5-O-(α-d-glucopyranosyl)-d-erythroascorbic acid and 5-methyl-5-O-(α-d-xylopyranosyl)-d-eryth-roascorbic acid were accumulated initially in the illuminated mycelia before the initiation of fruiting. The content of glycosides of erythroascorbic acid and their methylated compounds increased again in the primordia and the fruit bodies. Exogenous L-ascorbic acid induced the formation of primordia from the mycelia in the dark in a dose-dependent manner. Thus, this suggests that these reductones might play a role in mediating the light stimulus in photomorphogenesis.