DNA Polymorphisms in Lentinula edodes, the Shiitake Mushroom

DNA restriction fragment length polymorphisms (RFLPs) were examined in Lentinula edodes strains. Genomic DNA from strain 70 was cloned in plasmid vector pUC19, and 18 random clones containing low-copy DNA sequences were used to probe seven strains in Southern DNA-DNA hybridizations. Each cloned fragment revealed DNA polymorphism. An RFLP genotype was determined for each strain and the genetic relatedness was assessed. The coefficients of genetic similarity among the seven strains ranged from 0.43 to 0.90. The inheritance of RFLP markers was examined in single spore isolates. Homokaryons displayed a loss of polymorphic bands compared with the parent dikaryon. Hybrids constructed by crossing compatible homokaryons displayed the inheritance of RFLP markers from each parent homokaryon.     

一种含有单链RNA的香菇球状病毒

从生长不正常的香菇(Lentinus edodes(Berk.)Sing)菌株中分离到一种等轴对称含单链RNA的病毒颗粒。病毒颗粒在电镜下直径为33～34nm,在SDS-聚丙烯酰胺凝胶电泳中病毒外壳蛋白分子量为22000道尔顿。病毒核酸径DNase1和SI酶解试验及热变性紫外吸收曲线试验证明为单链RNA,在1.5％的琼脂糖凝胶电泳中,病毒核酸呈现一条带,分子量为2.38×10~6道尔顿。     

香菇栽培料新工艺

在国内香菇栽培料常规配方基础上,设计了添加2.5mL盐酸/kg取代常规配方中1%蔗糖的新工艺,经测定,2种栽培料灭菌后还原糖与蔗糖浓度、发菌期与子实体产量基本一致,新配方栽培料灭菌后pH值为5.6,是香菇菌丝生长最适pH值。新配方工艺可降低生产成本10%左右。     

Biodegradation of an Organoselenium Compound to Elemental Selenium by Lentinula edodes (Shiitake) Mushroom

The present paper reports for the first time the transformation of an organic selenium compound into red selenium (Se), which causes the intense red pigmentation of Lentinula edodes (shiitake mushroom) mycelia. The biotransformation of 1,5-diphenyl-3-selenopentanedione-1,5 (diacetophenonyl selenide, preparation DAPS-25) was studied in liquid- and solid-phase cultures of L. edodes. In liquid culture medium, a red color develops in the mycelium at initial DAPS-25 concentrations equal to or higher than 0.1?mmol/l. The intensity and initiation time of coloration is Se concentration-dependent. Semiquantitative data obtained by physicochemical methods on the extent of Se and acetophenone production suggest that L. edodes is able to absorb and/or destruct this organic Se xenobiotic.     

Extracellular Enzymes Produced by the Cultivated Mushroom Lentinus edodes during Degradation of a Lignocellulosic Medium

Although the commercially important mushroom Lentinus (= Lentinula) edodes (Berk.) Sing. can be rapidly cultivated on supplemented wood particles, fruiting is not reliable. This study addressed the problem by developing more information about growth and development on a practical oakwood-oatmeal medium. The study determined (i) the components degraded during a 150-day incubation at 22 degrees C, (ii) the apparent vegetative growth pattern, (iii) the likely growth-limiting nutrient, and (iv) assays that can be used to study key extracellular enzymes. All major components of the medium were degraded, lignin selectively so. The vegetative growth rate was most rapid during the initial 90 days, during which weight loss correlated with glucosamine accumulation (assayed after acid hydrolysis). The rate then slowed; in apparent preparation for fruiting, the cultures rapidly accumulated glucosamine (or its oligomer or polymer). Nitrogen was growth limiting. Certain enzyme activities were associated with the pattern of medium degradation, with growth, or with development. They included cellulolytic system enzymes, hemicellulases, the ligninolytic system, (gluco-)amylase, pectinase, acid protease, cell wall lytic enzymes (laminarinase, 1,4-beta-d-glucosidase, beta-N-acetyl-d-glucosaminidase, alpha-d-galactosidase, beta-d-mannosidase), acid phosphatase, and laccase. Enzyme activities over the 150-day incubation period with and without a fruiting stimulus are reported. These results provide a basis for future investigations into the physiology and biochemistry of growth and fruiting.     

PEG介导下香菇的转化

表达载体p301-bG1含有香菇（Lentinus edodes (Berk.)Sing)三磷酸甘油醛脱氢酶启动子驱动下的gus基因和除草剂抗性基因。利用PEG法实现了p301－bG1对香菇原生质体的转化。香菇原生质体与经PEG纯化的质粒DNA混合,用PEG处理后培养于含40ug/mL除草剂的CYM再生平板上,得到了抗除草剂和有GUS活性的转化菌株。虽然这种方法转化效率较低,但不需要昂贵的仪器和限制性内切酶,为蘑菇的分子育种研究提供了一种简便经济的转化方法。     

PEG-mediated Transformation of Lentinus edodes

Expression vector p301-bG1 contains a gus gene and a bialaphos resistance gene both driven by glyceraldehydes-3-phosphate dehydrogenase (GPD) gene promoter isolated from Lentinus edodes (Berk.) Sing. Using p301-bG1, PEG-mediated transformation of protoplast of L. edodes was studied. Mixed with PEG-purified plasmid DNA, the protoplasts of L. edodes were treated with PEG solution and cultured on CYM regeneration plate containing 40 μg/mL bialaphos. Bialaphos-resistant and GUS-positive transformants were obtained using this transformation system. Although the transformation efficiency was relatively low, the protocols release large expenses on expensive instrument and restriction enzymes, providing a simple and economical method for mushroom breeding at the molecular level.     

Lectin activity of Lentinus edodes

The hemagglutinating activity of submerged mycelium and culture liquid for four strains of Lentinus edodes (Berk.) Sing [L. edodes (Berk.) Pegler] was studied in the search for lectins. The hemagglutinating activity of culture liquid was substantially higher, compared with mycelium. The carbohydrate-binding capacity of the agglutinins was established, and the lectin activity of extracts from mycelia grown on several agar media was elucidated in relation to fruiting. The lectin activity of L. edodes was examined at different morphogenetic steps: mycelium, brown mycelial film, primordium, and fruiting body. Hemagglutination titers at the brown film step were higher than in the mycelium, whereas activity at the primordial and fruiting bodies steps decreased. Lectins seem to be involved in the formation of hyphal aggregates of brown mycelial film.     

Polyisoprenoid alcohols from the mushroom Lentinus edodes

Lipids extracted from the shiitake mushroom Lentinus edodes contain dolichols composed of 15 up to 19 isoprene units with Dol-17 as the dominating prenologue. Identification of dolichols was achieved by the application of 2D-TLC, HPLC and electrospray ionization mass spectrometry. Additionally a family of polyprenols (-unsaturated counterparts) with the same chain-length was also detected. Dolichols comprised approximately 0.002% of the fresh weight of the mushroom. Dolichols accompanied by traces of polyprenols are for the first time found in the mushroom tissue.     

The detection of single-stranded RNA in an isometric virus-like particle from Shiitake mushroom [Lentinus edodes (Berk.) Sing.]

Isometric virus-like particles (VLPs) with diameter of approximately 34 nm containing ss-RNA were purified from abnormal mycelium of Shiitake mushroom, Lentinus edodes (Berk.) Sing. SDS-polyacrylamide gel elec-trophoresis demonstrated that the virions contain a single capsid polypeptide with molecular weight of about 22 000 daltons. The nucleic acid extract from purified VLPs preparations showed only one band with a size of approximately 7.3 kilobases. The susceptibility to RNase 1 and S1 digestions, resistance to DNase and thermal denaturation behaviour of the viral genome indicated that it is a single-stranded RNA. To our knowledge, isometric single-stranded RNA VLPs isolated from Shiitake mushroom mycelia have not been reported before.     

Purification and characterization of a novel chitinase from Burkholderia cepacia strain KH2 isolated from the bed log of Lentinus edodes,Shiitake mushroom

One of the chitinases secreted in the culture filtrate of a gram-negative bacteria, Burkholderia cepacia strain KH2, which was isolated from the bed log of Lentinus edodes, Shiitake mushrooms, was purified by DEAE Sepharose CL-6B chromatography, followed by Sephacryl S-100 HR gel filtration. The purified enzyme was homogenous, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), with an estimated molecular weight of 34,000 and an isoelectric point (pI) of 5.9. The enzyme was stable at pH values of 4.0-6.0, and at temperatures up to 50 degrees C; the optimum pH and temperature were 4.5 and 50 degrees C, respectively. The enzyme exhibited higher activities toward chitosan 7B, a 62% deacetylated chitosan, than toward the highly deacetylated chitosan substrates. The enzyme was observed to drastically hydrolyze partially deacetylated chitin substrates, with the subsequent formation of N-acetylchitooligosaccharides [(GlcNAc) (n), n=2-7]. Separation and quantification of the hydrolysis products of (GlcNAc) (n), n52-6, by HPLC showed the splitting into (GlcNAc)(n), n=3-6. Activity toward N-acetylchitobiose was not detected. Oligomers with a higher number of units than the starting substrate were also detected, which indicate transglycosylation activity.     

Fruiting ofLentinus edodes (Shiitake) in liquid media

Summary Eight dikaryotic stocks ofLentinus edodes (Berk.) Sing. were studied in respect to fruiting in several different formulations of chemically-defined liquid media. On the two media which included the same four solutions (minerals, trace elements, vitamins and salicylic acid) the stocks differed only slightly in growth but were markedly different in primordia and fruitingbody formation. Stocks also showed variation in respect to fruiting, following incubation in media from which one of the solutions was omitted, and it was suggested that this was a consequence of differences in genes controlling fruiting. Inorganic substances (glass wool, vermiculite and perlite) served satisfactorily in supporting fruiting bodies in liquid media. Light was essential for borwn-pigment formation of the mycelial coat and for fruiting-body maturation but was not required for formation of primordia.

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Resumen Se estudío la fructificación de ocho cepas dikaríoticas de Lentinus edodes en varias fórmulas químicas diferentes de medio líquido definido. En dos medios que contenían las mismas cuatro soluciones (mineral, elementos trazas, vitaminas y ácidos salicílico) las diferencias en crecimiento entre las cepas eran apenas perceptibles pero eran marcadamente diferentes en relación a la formación del primordio y del cuerpo fructífero. Las cepas también mostraban variación en la fructificatión cuando se incubaban en un medio al cual una de las soluciones se le omitía, sugiriéndose ésto como una consecuencia de diferencias en los genes controlando la fructificación. Sustancias inorgánicas (lana de vidrio, vermiculita y perlita) fueron usadas satisfactoriamente como soporte de los cuerpos fructíferos en medio líquido. La luz era esencial para la formación del pigmento marrón de la capa micelial y para la maduración del cuerpo fructífero, pero no era requerida para la formación de primordio. Received 1 November 1986; accepted 29 December 1986

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Résumé Huit souches dicaryotes deLentinus edodes (Berk.) ont été étudiées en ce qui concerne la fruitification dans différentes formulations de milieux liquides chimiquement définis. Sur les deux milieux contentant les quatre mêmes solutions (composés minéraux, oligo-éléments, vitamines et acide salicylique), les souches différent peu en ce qui concerne la croissance, mais par contre foretement en ce qui concerne la formation de primordia et de fructifications. La fructification des souches varie aussi lorsque l'incubation est effectuée dans des milieux où l'une gènes contrôlant la frucitification. Certaines substances minérales (laine de verre, vermiculite et perlite) soutiennent efficacement les fructifications en mlieu liquide. La lumière est nécessaire à la pigmentation brune du mycélium et à la maturation des fructifications, mais n'influe pas sur la production de primordia.

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Visiting Professor in the Department of Biology, CUHK, from the State University of New York at Buffalo, USA.     

Tolerance of tannin by the shiitake mushroom,Lentinus edodes

Summary Tannin at 1% (w/v) did not inhibit the growth ofLentinus edodes, but did inhibitPleuroius florida, P. sajor-caju, P. cystidosus, Agaricus bisporus andVolvariella volvacea. The inhibition was not due to its acidity.

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Tolérance de Lentinus edodes aux ajouts de tannin

Résumé Le tannin à la concentration de 1% (p/v) n'inhibe pas la croissance deLentinus edodes, mais inhibe celle dePleurotus florida, P. sajor-caju, P. cystidosus, Agaricus bisporus, etVolvariella volvacea. L'inhibition n'est pas due à son acidité.

     

香菇双单杂交后代不同发育阶段酯酶同工酶研究

选用香菇野生株分别与栽培株及杂交株进行双单杂交 ,得到 8个杂交后代 ,且具有结实能力 ,分别对液培 2 0d杂交后代菌丝与液培原基酯酶同工酶进行了比较研究。结果表明 ,液培 2 0d菌丝菌株间酯酶同工酶酶谱显示出多样性 ,可以作为鉴定菌株的辅助遗传标记 ,而原基菌株间酯酶同工酶酶谱谱带较少 ,呈现趋同效应 ,不宜作为鉴定香菇菌株的辅助遗传标记。     

Substrate specificity of laccase from Lentinus edodes

In previous studies, the white-rot basidiomycete Lentinus edodes, strain SC-495, was proved to be a “selective” lignin degrader and its extracellular crude preparations arising from solid-state cultures were successfully employed in biopulping experiments on annual plants. This fungus produced extracellular laccase as the predominant phenoloxidase when growing in solid-state fermentation on corn stalks. Laccase from this strain was purified and partially characterized, as an initial approach towards the study of its ligninolytic complex. Laccase was purified 69.6-fold by anion-exchange chromatography and two affinity-chromatography steps with an overall yield of 7.45%. The native enzyme exhibited a molecular mass of 74 kDa, an isoelectric point of 3.42 and a carbohydrate content of 7.5%. The absorption spectrum of laccase showed a maximum at 605 nm, typical of blue-copper oxidases. The optimum pH and temperature for the activity of laccase were 4.0–4.2 and 50°C, respectively. Kinetic experiments, performed with a wide range of phenolic compounds, showed that the reaction rate and the substrate affinity greatly varied depending on the nature of substituents and their reciprocal positions on the aromatic ring. In particular, the enzyme showed high affinity to phenolic compounds bearing methoxyl or methyl groups, but no affinity to those bearing the nitro group directly attached to the benzene ring, nor to non-phenolic lignin-related compounds, such as trans-cinnamic acid or 3,4-dimethoxycinnamic acid. The huge differences in terms of reactivity of the enzyme towards phenolic compounds suggests that a preliminary systematic screening should be advisable when using laccase in effluent treatment applications.     

Cultivation of the Dible Mushroom Lentinula edodes (Shiitake) in Pasteurized Wheat Straw – Alternative Use of Georthermal Energy in Mexico

Five edible Lentinula edodes strains were evaluated. The mushrooms were cultivated on a wheat straw substrate that was previously pasteurized by immersion in water heated by residual geothermal vapor, which was also used to warm incubation and production rooms. Finely chopped wheat straw (Triticum aestivum L.) was pasteurized and then spawned with supplemented spawn capable of supplying nutrients and enriching the substrate, with the expectation of yield improvement. The samples were incubated for 60 days before the production started and thus, the mushrooms produced had pileus diameters ranging from 5 to 20 cm. The yields fluctuated from 6.2 to 13.9 % (fresh weight of mushrooms/fresh weight of substrate). Biological efficiency ranged from 24.8 to 55.6 % (fresh weight of mushrooms/dry weight of substrate), while the production rate reached varied from 0.19 to 0.55 % (biological efficiency/production time starting from inoculation). The cultivation system evaluated here offers the possibility of lowering production costs by cultivating the mushroom on easily obtainable substrate and shortening the culture cycle. The efficiency of this use of geothermal energy and supplemented spawn for shiitake mushroom cultivation on non‐sterilized substrates was proven.     

Salt-Regulated Mannitol Metabolism in Algae

Mannitol, one of the most widely occurring sugar alcohol compounds, is found in bacteria, fungi, algae, and plants. In these organisms the compound acts as a compatible solute and has multiple functions, including osmoregulation, storage, and regeneration of reducing power, and scavenging of active oxygen species. Because of the diverse functions of mannitol, introducing the ability to accumulate it has been a hallmark of attempts to generate highly salt-tolerant transgenic plants. However, transgenic plants have not yet improved significantly in their salt tolerance. Recently, we purified and characterized 2 enzymes that biosynthesize mannitol, mannitol-1-phosphate dehydrogenase (M1PDH) and mannitol-1-phosphate-specific phosphatase, from the marine red alga Caloglossa continua, which grows in estuarine areas where tide levels fluctuate frequently. The activation of Caloglossa M1PDH is unique in that it is regulated by salt concentration at enzyme level. In this review we focus on the metabolism of mannitol, mainly in marine photosynthetic organisms, and suggest how this might be applied to producing salt-tolerant transgenic plants.     

Analysis of mfbA alleles of the eubasidiomycete Lentinus edodes

A fruiting-body-specific mfbA cDNA derived from Lentinus edodes FMC2 has been shown to encode a high-molecular-weight protein, MFBA, containing the cell-adhesion-promoting Arg-Gly-Asp (RGD) sequence. Southern-blot analysis showed that all L. edodes strains tested have the mfbA gene (homologue). Nucleotide sequence analysis of the 1-kb mfbA fragments containing the RGD-coding sequence showed that each L. edodes strain has two types of mfbA homologues. It was found in FMC2 that two mfbA homologues are derived from different nuclei and these mfbA alleles are transcribed with similar frequencies in the fruiting bodies.