Isolation and characterization of the gene encoding a chitin synthase with a myosin motor-like domain from the edible basidiomycetous mushroom, <Emphasis Type="Italic">Lentinula edodes</Emphasis>, and its expression in the course of fruit-body formation

We isolated and characterized the genomic and complementary DNAs encoding a chitin synthase from an edible basidiomycetous mushroom, Lentinula edodes. The gene (which we designated Lechs1) contains a large open reading frame encoding a polypeptide of 1937 amino acid residues. The open reading frame is interrupted by 14 small introns (49–116 bp). The gene product (LeChs1) consists of a myosin motor-like domain in its N-terminal half and a chitin synthase domain in its C-terminal half, analogous to the class V and VI chitin synthases of other filamentous fungi. Phylogenetic analysis demonstrated that LeChs1 is classified into class VI chitin synthases. Southern blot analysis indicated that Lechs1 is a single-copy gene per haploid genome and that L. edodes has no other highly homologous chitin synthase genes. Northern blot analysis revealed that Lechs1 is expressed throughout the whole stages of fruit-body formation of L. edodes, but its expression level gradually declines in a fruit body-maturation-dependent manner with highest expression in vegetative mycelia and fruit body at the early stage of maturation (immature fruit body). This is the first report on the isolation and characterization of the gene encoding a chitin synthase with a myosin motor-like domain from basidiomycetes.     

Mycelial yield of<Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> using thinned apple,pear, and peach on submerged culture

The effect of thinned fruits, apple, pear and peach, on the mycelial growth of mushrooms was investigated. The growth of mycelia with the addition of thinned fruit was clearly better than that in the control for all the tested mushrooms. The growth rate ofPleurotus ostreatus was faster than any other mushroom. The optimal concentrations of thinned apple, pear, and peach in a solid culture were 1.0%, 1.0%, and 3%, respectively, while in a liquid culture the optimal concentrations were 5,0%, 3.0%, and 5.0%, respectively. WhenPleurotus ostreatus was incubated in a 20-L pilot scale fermenter with 10 L of a liquid medium containing 3% thinned fruit at 25°C and 6 vvm for 10 days, the mass-production of mycelia was 74.2 g/10 L (apple), 96.2 g/10 L (pear), and 86.3 g/10 L (peach). The mycelial yield ofPleurotus ostreatus in a medium containing thinned fruit was 2≈3 times higher than that in the control.     

Fruit-body production of an ectomycorrhizal fungus in genus <Emphasis Type="Italic">Boletus</Emphasis> in pure culture

Mycelial growth and fruit-body production of an ectomycorrhizal Boletus sp. were examined in pure culture. Mycelia of the strain Bo1 grew well on a medium consisting of sawdust and barley grains. Mature fruit bodies bearing basidiospores were produced after incubation at 22°C for 90 days in the dark, followed by incubation at 26°C for 30–46 days under conditions of high humidity and illumination. The addition of porous stone as a casing on the medium increased fruit-body yield. Deposited spores germinated well on an agar medium and formed mycelial colonies, thus completing the life cycle of Bo1 without a host plant and under axenic conditions. The ability of Bo1 to form ectomycorrhizas was confirmed by axenic resynthesis of mycorrhizas on Quercus serrata. Cultured fruit bodies of Bo1 resembled Gyroporus castaneus and Boletus subcinnamomeus, but its taxonomic position was not elucidated at the species level.     

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     

A possible role of the key enzymes of the glyoxylate and gluconeogenesis pathways for fruit-body formation of the wood-rotting basidiomycete Flammulina velutipes

 Biochemical roles of the representative enzymes involved in carbon metabolism of glucose were investigated in relation to the fruit-body formation of the basidiomycete Flammulina velutipes. Changes in specific activities of the enzymes of the tricarboxylic acid (TCA) cycle and glyoxylate (GLOX) and gluconeogenesis pathways were measured at different stages of development of the fungus. The enzyme activities of malate synthase (MS) and fructose bisphosphatase (FBP) as the key enzymes for the GLOX-gluconeogenesis pathways increased in mycelia during the fruit-body formation. The activities of isocitrate dehydrogenase (IDH) for the TCA cycle and NADP-linked glutamate dehydrogenase (GLTDH (NADP)) for glutamate synthesis increased more markedly. Moreover, the mycelial mat of the cultures producing fruit bodies yielded greater enzyme activities of isocitrate lyase (ICL), MS, FBP, and IDH than that of the cultures that did not produce fruit bodies. These results suggest that the GLOX-gluconeogenesis pathways as well as the glutamate synthesis have a strong correlation with the fruit-body formation of F. velutipes. Received: January 22, 2002 / Accepted: May 10, 2002     

DNA marking of some quantitative trait loci in the cultivated edible mushroom <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> (Fr.) Kumm.

Fungi of the genus Pleurotus, in particular, species Pleurotus ostreatus (common oyster mushroom) are among most cultivated fungi in the world. Due to intense rates of development of studies in this field, efficient breeding programs are highly required in the search for new P. ostreatus strains. The principal traits used worldwide for selecting strains are intensity of fruitbearing, fruit body cap color (for some consumptive markets), and mycelium growth rate. In this connection, the objective of this work was to study these quantitative traits and to find molecular markers, which could be employed to accomplish breeding programs. In general, we found 12 genomic loci (quantitative trait loci, QTLs) controlling mycelium growth rate of oyster and six QTLs responsible for the fruit body cap color. The genetic map of P. ostreatus was constructed, and all markers of quantitative traits found by us were located on this genetic map. The obtained linkage map can be a useful tool for the accomplishment of breeding programs to improve economically important traits of oyster 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.     

Isolation of a gram-positive bacterium effective in suppression of brown blotch disease of cultivated mushrooms,Pleurotus ostreatus andAgaricus bisporus, caused byPseudomonas tolaasii

A Gram-positive bacterium was isolated from a rottingPleurotus ostreatus fruiting body that markedly reduced the level of extracellular toxins (i.e., tolaasins) produced byPseudomonas tolaasii, the most destructive pathogen of cultivated mushrooms. The isolated bacterium is saprophytic but not parasitic nor pathogenic toP. ostreatus. A low ratio, ca. 10⁻³ cells of the isolated bacterium for oneP. tolaasii cells, was sufficient for detoxification in vitro. Inoculation of the isolated bacterium prevents the development of bacterial disease inP. ostreatus andAgaricus bisporus. The suppression of the disease development, however requires the initial cell density equivalent to ca. 10⁻¹ cells of the isolated bacterium for one cells of the pathogen. The effects is ascribed to the inactivation of tolaasin by the live, suppressive bacterial cells, and not to metabolites secreted from the organism into culture media. Examination by conventional bacteriological tests and with testing kits, i.e., MicroStation^TMSystem Release 3.5 (Biolog Inc., Hayward, CA), ATB Expression (bioMerieux Inc. Japan) and VITEK (bioMerieux Inc. Japan), failed to assign the organism to any defined bacterial genus. The suppressive bacterium may be useful in future for the development of biocontrol system and/or the construction of genetically modified edible fungi resistant to the disease caused byP. tolaasii.     

Changes in low molecular weight carbohydrates composition and chitin throughout the cultivation stages ofPleurotus ostreatus

Changes in contents of soluble low molecular weight carbohydrates and chitin in a sawdust-rice bran medium during mycelial growth ofPleurotus ostreatus in bottle cultivation were examined in relation to fruit-body yield of nine stocks. Glucose, mannitol, inositol, sucrose, and trehalose were detected in cultures after mycelial spreading. No significant correlation was observed between contents of soluble low molecular weight carbohydrate during mycelial growth and the fruit-body yield. Negative correlation was found between trehalose content in post-harvest cultures and the fruit-body yield. Chitin content in cultures decreased in the fruiting stage. Positive correlation was detected between chitin content of fruit-bodies and the decrement of chitin in post-harvest culture caused by fruit-body growth.     

Response Surface Analysis of Solid State Growth of <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> Mycelia utilizing Whey Permeate

A novel approach to utilizing whey permeate, the cultivation of mycelia of the edible mushroom Pleurotus ostreatus, is introduced. Response surface analysis (RSA) was successfully applied to determine the combination of substrate concentration, temperature and pH that would result in a maximal mycelial extension rate under solid state cultivation. The conditions to maximize the mycelial extension rate were predicted to be 44 g lactose l⁻¹, pH 6.0 and 24.2 °C. Subsequent verification of these levels agreed with model predictions.     

EXPERIMENTAL CONTROL OF FRUIT-BODY FORMATION IN COPRINUS MACRORHIZUS

In order to establish a proper experimental system for studying the physiological and biochemical processes of fruit-body formation in a Basidiomycetes, Coprinus macrorhizus Rea f. microsporus Hongo, various culture conditions, especially light and temperature, were investigated. The use of homogenates of fruit-body primordia, instead of mycelia, as inocula was effective for maintaining cultures capable of active fruit-body formation during many successive cultivations. At least two successive intermittent illuminations every 12 hr during the 3rd and 4th days of incubation were required for the differentiation of pilei. The most effective wave lengths of incident light were found in the region from 440 to 485 mμ. Temperature effects on fruit-body formation were exhibited in two stages of morphogenesis; i.e., in the induction of primordia formation from vegetative hyphae (by 5°-treatment) and in the transition phase from primordia formation to fruit-body development (by 20°-treatment). Based on these observations, three experimental systems for physiological and biochemical studies on the fruit-body formation have been proposed.     

Effect of oxygen concentrations and branched-chain amino acids on the growth and development of sub-seafloor fungus,Schizophyllum commune 20R-7-F01

Fungi have been reported to be the dominant eukaryotic group in anoxic sub-seafloor sediments, but how fungi subsist in the anoxic sub-marine sedimental environment is rarely understood. Our previous study demonstrated that the fungus, Schizophyllum commune 20R-7-F01 isolated from a ~2 km sediment below the seafloor, can grow and produce primordia in the complete absence of oxygen with enhanced production of branched-chain amino acids (BCAAs), but the primordia cannot be developed into fruit bodies without oxygen. Here, we present the individual and synergistic effects of oxygen and BCAAs on the fruit-body development of this strain. It was found that the fungus required a minimum oxygen concentration of 0.5% pO₂ to generate primordia and 1% pO₂ to convert primordia into mature fruit body. However, if BCAAs (20 mM) were added to the medium, the primordium could be developed into fruit body at a lower oxygen concentration up to 0.5% pO₂ where genes fst4 and c2h2 playing an important role in compensating oxygen deficiency. Moreover, under hypoxic conditions, the fungus showed an increase in mitochondrial number and initiation of auto-phagocytosis. These findings suggest that the fruit-body formation of S. commune may have multiple mechanisms, including energy and amino acid metabolism in response to oxygen concentrations.     

Saponin Stimulates Fruiting of the Edible Basidiomycete Pleurotus ostreatus

The addition of saponin from Quillaja to malt extract agar dramatically stimulated fruit body development of Pleurotus ostreatus. Higher concentration of Quillaja saponin added to the medium suppressed the growth of pilei of fruit bodies. The results indicate that the triterpenoid-saponin is a bioactive substance for fruiting of P. ostreatus.     

Effects of different nitrogen rich substrates and their combination to the yield performance of oyster mushroom (<Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>)

This study was conducted to assess the effect of nitrogen (N) variations from lignocellulosic substrates of empty fruit bunch (EFB), palm oil frond (POF), palm oil trunks (POT), A.mangium bark (AB) and Cogongrass (IC), and combinations among them using biological supplements (POF and POT) for suitability as a substrate of P. ostreatus. Three different sets of substrates were evaluated in response to different parameters of P. ostreatus including spawn running, primordial initiation, fruit body yield and biological efficiency (BE). The supplementation of POT and POF generally revealed significant differences on the crop performance of P. ostreatus compared to the un-supplemented substrate. POT demonstrated reduced production time, while POF was the best supplement to promote fresh weight and BE. The most rapid spawn running took place within 18.6 ± 0.9 days, while the primordial initiation was observed within 23.2 ± 0.5 days in the combination of IC + POT. The most suitable substrate for higher mushroom weight and BE was IC + POF (19.6 ± 1.3 g; 70.5 ± 5.5%). In addition, the interval between primordial formations was found to be fastest in the un-supplemented EFB and the combination of EFB + POF, which supported a shortened production time. Therefore, IC and EFB appeared to be the better alternative materials for growing P. ostreatus, either with or without any supplementation.     

大蒜浸出液对平菇细菌性褐斑病病原菌托拉斯假单胞杆菌的抑菌作用

【背景】由托拉斯假单胞杆菌(Pseudomonas tolaasii)引起的平菇细菌性褐斑病在国内外大面积发生,导致产量降低,并有潜在的安全风险,寻找安全有效的抑菌剂对产业发展具有重要意义。【目的】通过5种不同溶剂提取得到大蒜浸出液,测定其对平菇细菌性褐斑病病原菌托拉斯假单胞杆菌的抑制作用,同时检测其对平菇菌丝生长的作用。【方法】利用抑菌圈法测定5种不同的大蒜浸出液对托拉斯假单胞杆菌的抑菌作用,利用平板扩散法筛选能促进平菇菌丝生长的药剂及适宜的浓度。【结果】5种大蒜浸出液原液对托拉斯假单胞杆菌均有显著的抑菌活性,其中大蒜山杏壳木醋液浸出原液抑菌效果最强。不同浓度的大蒜浸出液抑菌作用比较发现,浓度为10%的大蒜山杏壳木醋液浸出液具有较好的抑菌效果,其抑菌效果与0.33 mg/mL的链霉素相当,并对平菇菌丝生长有显著的促进作用,菌丝生长速度显著大于对照,并且菌丝浓密,边缘整齐。【结论】本研究为大蒜与山杏壳木醋液复配药剂防治平菇细菌性褐斑病奠定了实验基础。     

rtpA, a gene encoding a bacterial two-component sensor kinase, determines pathogenic traits ofPseudomonas tolaasii, the causal agent of brown blotch disease of a cultivated mushroom,Pleurotus ostreatus

Pseudomonas tolaasii strain PT814 produces extracellular toxins, tolaasins, and a volatile toxin, tovsin, that are responsible for the induction of brown blotch and rotting, respectively, in a cultivated mushroom,Pleurotus ostreatus. Insertions of single transposon mini-Tn5Km 1 into the chromosome ofP. tolaasii strain PT814 generated mutants that are pleiotropically defective in tolaasin and protease production, and altered in colony morphology. The mutants, however, produce tovsin at the level of wild-type. Variants phenotypically similar to the pleiotropic mutants ofP. tolaasii strain PT814 spontaneously occurred inP. tolaasii strain S8501 at 22–30°C in vitro. The occurrence of variants was significantly reduced in the presence of extracts ofP ostreatus or at a temperature of 15–20°C. ThertpA gene (rtpA=regulator gene of tolaasin production and other pleiotropic traits) isolated from aP. tolaasii strain PT814 gene library restored the wild-type phenotype in both the mini-Tn5km 1 insertion and spontaneous mutants. mini-Tn5km 1 insertions were also located in the allele ofrtpA. Nucleotide sequencing of thertpA DNA revealed an open reading frame of 2,751 bp predicted to encode a protein consisting of 917 amino acid residues with a molecular mass of 100.6 kDa and displaying the conserved amino acid sequence of both sensor, and receiver domains of “bacterial two-component regulators”. The data suggest that the machinery responding to environmental stimuli is essential for the pathogenic interaction ofP. tolaasii with the mushroom.     

Metabolic function of glycogen phosphorylase and trehalose phosphorylase in fruit-body formation ofFlammulina velutipes

Glycogen phosphorylase in the vegetative mycelium ofFlammulina velutipes converts glycogen to α-glucose 1-phosphate (G1P) in the colony during fruit-body development. Glycogen may contribute to the synthesis of trehalose as the starting material in the vegetative mycelium during the fruiting process of the colony, and the trehalose produced is translocated into the fruit-bodies as the main carbohydrate substrate for their development. Trehalose phosphorylase activity in the vegetative mycelium was at a relatively high level until fruit-body initiation, suggesting the turnover of this disaccharide during the vegetative stage of the colony development. Trehalose phosphorylase activity in the stipes showed a peak level at the early phase of fruit-body development, suggesting the continuing phosphorolysis of trehalose by this enzyme. The stipes also showed a high specific activity of phosphoglucomutase at a sufficient level to facilitate the conversion of G1P to α-glucose 6-phosphate (G6P). In the pilei a large amount of G1P remained until the growth of the fruit-bodies ceased. Trehalase activities in the stipes and pilei were at a very low level, and this enzyme may not contribute to the catabolism of trehalose in the fruit-body development.     

Cultivation of oyster mushrooms (<Emphasis Type="Italic">Pleurotus</Emphasis> spp.) on various lignocellulosic wastes

Cultivation of speciality mushrooms on lignocellulosic wastes represents one of the most economically and cost-effective organic recycling processes. Three species of Pleurotus, namely P. columbinus, P. sajor-caju and P. ostreatus were experimentally evaluated on untreated organic wastes including chopped office papers, cardboard, sawdust and plant fibres. Production studies were carried out in polyethylene bags of about 1 kg wet weight with 5% spawning rates of substrate fresh weight in a custom-made growth room especially designed for spawn run and cropping. The conversion percentage from dry substrate weight to fresh mushroom weight (biological efficiency) was determined. The highest biological efficiency was noted with P. columbinuson cardboard (134.5%) and paper (100.8%), whereas P. ostreatus produced maximum yield on cardboard (117.5%) followed by paper (112.4%). The overall yield of P. sajor-cajuwas comparatively low (range 47–78.4%). The average number of sporophore flushings ranged between 5 and 6 times. The findings that P. columbinus and P. ostreatus are superior to P. sajor-caju are consistent with previous reports elsewhere. Further evaluation of P. columbinus alone on different bagging systems containing partially pasteurized office papers as a growing substrate revealed that polyethylene bags resulted in 109.4% biological efficiency in contrast to pottery (86%), plastic trays (72%) or polyester net (56%). The above findings reveal an opportunity for commercial implication of oyster mushroom especially P. columbinus for utilization of different feasible and cheap recyclable residues.     

Lipogenesis in the Basidiomycetes Pleurotus ostreatusand Flammulina velutipes Cultivated on Different Media

The compositions of free fatty acids (FA) in the mycelia of oyster cap (Pleurotus ostreatus (Jacq. ex Fr.) Kumm.) and honey mushroom (Flammulina velutipes (Curt. ex Fr.) Sing.) and the effect of mycelium cultivation conditions on the composition and proportions of individual FA were investigated. Palmitic and linoleic acids were found to be major acids produced byP. ostreatus growing on solid agar medium and in a submerged culture with a synthetic medium. The composition of minor FA in P. ostreatuswas dependent on cultivation conditions. Surface cultivation of its mycelium yielded pentadecanoic, octadecenoic, and stearic acids. Submerged cultivation additionally yielded undecanoic, myristic, hexadecenoic, and lignoceric acids. The composition of free FA in F. velutipesshowed no significant differences from that of P. ostreatus. Variation in the C/N ratio in the cultivation medium affected both the FA composition in P. ostreatus and F. velutipes and the relationship between saturated and unsaturated FA.