Analysis of expressed sequence tags (ESTs) from Lentinula edodes

The 1,031 expressed sequence tags (ESTs) from the basidiomycete Lentinula edodes were generated as a pilot experiment to see distribution of genes expressed in L. edodes. Among them, genes for hydrophobin, which are specifically found in filamentous fungi, were the most frequently obtained ESTs (33 times), suggesting that they are highly expressed in L. edodes. In addition to known hydrophobin 1 and 2 types, our analysis revealed the existence of novel types of hydrophobin, which we named hydrophobin 3, 4, and 5. The second and the third most highly obtained ESTs were phosphatidylserine decarboxylase and formate dehydrogenase, which were obtained eight and seven times, respectively. It should be noted that two important genes (argonaute and RNA-dependent RNA polymerase) involved in the RNAi pathway were found, suggesting a future application for gene knock-down by RNA interference. The 53 ESTs were identical with the sequences already reported in L. edodes. The 433 ESTs were found to show significant sequence similarity (E value <1 x 10(-5)) with the proteins reported (or predicted) in other species. In total, 387,952 bp were sequenced and registered in DDBJ/GenBank (accession number BJ998097-BJ999127).     

Effect of liquid mycelial culture used as a spawn on sawdust cultivation of shiitake (Lentinula edodes)

Mycelium of shiitake (Lentinula edodes) was cultured continuously in liquid medium. The liquid culture was carried out for the production of liquid spawn in the cultivation of this mushroom on synthetic sawdust substrate, and its performance was compared with that of the solid spawn. The initial colonization in culture bags was faster with the solid spawn than with the liquid spawn, but after this stage CO₂ production was higher with the liquid spawn than with the solid spawn. For harvesting sufficient amount of good quality mushrooms, 120 d of incubation in bags was needed with the solid spawn, but this was reduced to 90 d for the sawdust blocks using liquid spawn of less than 50 d old. If continuous culture of the liquid spawn was prolonged over 50 d, immature fruit-bodies or their initials formed during the period of bag incubation. The solid subcultures of the liquid spawns retained the fruiting characteristics acquired in the liquid culture. Liquid culture could be a useful tool for breeding of mushrooms.     

Detection and distribution of six linear mitochondrial plasmids in the shiitake mushroom,Lentinula edodes

The presence of plasmids was surveyed in 90 wild isolates ofLentinula edodes collected from geographically different world regions. DNA plasmids of different sizes were found in about 80% of the isolates. The plasmids detected were of six kinds, designated as pLE1 (9.0 kb), pLE2 (11.1 kb,=pLLE1 described by other authors), pLE3A (9.8 kb), pLE3B (10.8 kb), pLE3C (12.1 kb), and pLE3D (12.3 kb). Hybridization analysis suggested that pLE1 and pLE2 were distinct plasmid types of different homology groups to each other, and the four other plasmids were variant types belonging to a third homology group. These plasmids had no homology with their host's and non-host's nuclear and mitochondrial genome DNAs. Restriction analysis and electron microscopy indicated that the plasmids are linear in form. Since all six plasmids were transmitted uniparentally in sexual crosses and were consistently associated with the DNA preparations from mitochondria fractionated from mycelia of representative isolates, they were suggested to be located in mitochondria, similar to many other known fungal DNA plasmids. Geographically, pLE1 and pLE2 were widely distributed in natural populations ofL. edodes, while the remaining four plasmids were uniquely present in delimited natural populations. Contribution No. 322 from the Tottori Mycological Institute.     

A new biological function of Shiitake mushroom,Lentinula edodes, in a myco-heterotrophic orchid,Erythrorchis ochobiensis

The biological function ofLentinula edodes in a myco-heterotrophic orchid,Erythrorchis ochobiensis was examined, using one local variant each from Japan (JPN), Papua New Guinea (PNG) and New Zealand (NZ). All variants induced seed germination: PNG and NZ isolates were effective at 25°C and JPN isolate showed the highest germination rate at 30°C. Germinated seeds developed into plants and formed normal endomycorrhizas. Hence, it is concluded thatL. edodes has a perfect symbiotic potential withE. ochobiensis, though it has not been observed in the root of the orchid in the field.     

Genetic analysis of nuclear ribosomal DNA of Lentinula edodes

Genetic analysis of nuclear ribosomal DNA (rDNA) of Lentinula edodes was carried out using rDNA restriction fragment length polymorphisms (RFLPs) as genetic markers. Two compatible monokaryotic strains that differed in the endonuclease digestion patterns of their rDNA were used. The dikaryotic strain established by crossing them produced mixed RFLP patterns. Single-spore isolates derived from the dikaryotic strain showed three types of rDNA RFLP patterns: either one of the two parental types or a mixed type. From the frequency of the mixed type, the recombination value of rDNA tandem repeats was calculated to be 31.4%. Linkage analysis between rDNA and two incompatibility factors (A and B) revealed that rDNA was not linked to either factor. The rDNA genotypes did not affect mycelial growth among the single-spore isolates.     

Characterization of Active Lentinula edodes Glucoamylase Expressed and Secreted by Saccharomyces cerevisiae

The gene encoding Lentinula edodes glucoamylase (GLA) was cloned into Saccharomyces cerevisiae, expressed constitutively and secreted in an active form. The enzyme was purified to homogeneity by (NH₄)₂SO₄ fractionation, anion exchange and affinity chromatography. The protein had a correct N-terminal sequence of WAQSSVIDAYVAS, indicating that the signal peptide was efficiently cleaved. The recombinant enzyme was glycosylated with a 2.4% carbohydrate content. It had a pH optimum of 4.6 and a pH 3.4–6.4 stability range. The temperature optimum was 50°C with stability ≤50°C. The enzyme showed considerable loss of activity when incubated with glucose (44%), glucosamine (68%), galactose (22%), and xylose (64%). The addition of Mn⁺⁺ activated the enzyme by 45%, while Li⁺, Zn⁺⁺, Mg⁺⁺, Cu⁺, Ca⁺⁺, and EDTA had no effect. The enzyme hydrolyzed amylopectin at rates 1.5 and 8.0 times that of soluble starch and amylose, respectively. Soluble starch was hydrolyzed 16 and 29 times faster than wheat and corn starch granules, respectively, with the hydrolysis of starch granules using 10× the amount of GLA. Apparent K_m and V_max for soluble starch were estimated to be 3.0 mg/ml and 0.13 mg/ml/min (40°C, pH 5.3), with an apparent k_cat of 2.9×10⁵ min⁻¹.     

Morphological mutation of Lentinula edodes mycelium, particularly detectable in the dikaryotic state

A morphological mutation particularly detectable in the dikaryotic state was found in Lentinula edodes. The mutant dikaryon was readily distinguishable from the normal dikaryon by the irregularly branched short hyphae, very slow hyphal growth, and sparse aerial hyphae. Genetic analysis revealed that expression of this mutation was controlled by a single recessive gene, mor-13. Linkage analysis showed that the mor-13 was not linked to either the incompatibility factors (A and B) or the five kinds of mor genes that were segregated independently of each other in a previous study. Contribution no. 380 from the Tottori Mycological Institute     

Purification and characterization of an extracellular acid trehalase fromLentinula edodes

Trehalase from the culture filtrate ofLentinula edodes was purified and characterized. Molecular masses were estimated to be 158 kDa and 79–91 kDa by gel filtration and SDS-PAGE under the reduced condition, respectively. The enzyme was composed of two identical subunits and contained carbohydrate molecules. The optimum temperature and pH were obtained at around 40°C and pH 5.0, respectively. The enzyme was stable up to 40°C and in a range pH of 4–10 at 30°C. It cleaved α-1,1 linkages of trehalose, but not α-1,4, α-1,6 or β-1,4 glycosyl linkages, and was defined as an acid trehalase.     

Induction of apoptosis in cultured cells by extracts from shiitake (Lentinula edodes) mycelial culture broth

Extracts fromshiitake (Lentinula edodes) mycelial culture broth, by an organic solvent ethyl acetate, inhibited the proliferation of cultured cells. At lower concentrations (1.25–15 μg/ml), this inhibition, measured by the MTT assay, was dose- and cell line-dependent. Inhibition of tumor cells, such as Caski, SiHa, HeLa, HP-1 and A375, byL. edodes-436 extracts was stronger than inhibition of normal cells (3T3). At 20 μg/ml, the extracts induced changes in cell shape, DNA-fragmentation and the activation of caspase-3. The extracts also inhibited the binding of E2F protein to its promoter. The results suggest that extracts ofL. edodes culture broth contain substances that have the ability to induce apoptosis in the cultured cells.     

A simple, rapid and effective method for total RNA extraction from Lentinula edodes

A rapid, inexpensive and reliable method for total RNA extraction from fruiting bodies of Lentinula edodes containing large quantities of polysaccharides and secondary metabolites is described. An initial extraction step using saturated NaCl solution facilitates the separation of nucleic acids from contaminants and, after further extraction with organic solvents and precipitation with 2-propanol, total RNA of high purity and suitable for applications such as cDNA synthesis, RT-PCR and Northern blot hybridization was obtained. The procedure may also have wider applicability for total RNA extraction from the tissues of other mushrooms.     

Differential scanning calorimetric analysis of fruit-body and mycelium ofLentinula edodes

Differential scanning calorimetric (DSC) analysis was applied to the fruit-body (pileus and stipe) and mycelium, of shiitake mushroom (Lentinula edodes). Thermograms of each sample indicated distinctive patterns. However, chemical and infrared (IR) spectroscopic analyses showed that the compositions of pileus and stipe were similar to each other and different from that of mycelium. Because the DSC thermogram depends not only on chemical composition but also on physical properties such as density, the result of DSC analysis was assumed to indicate a difference in the state of cell wall between pileus and stipe.     

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.     

Purification of a novel extracellular laccase from solid-state culture of the edible mushroom <Emphasis Type="Italic">Lentinula edodes</Emphasis>

The laccases (EC 1.10.3.2) secreted into solid-state culture by Lentinula edodes were analyzed. The fungus secreted at least two laccases in the solid-state culture. One laccase was purified to a homogeneous preparation using anion-exchange, hydrophobic, and size-exclusion chromatography. SDS-PAGE analysis showed that the purified laccase, Lcc6, was a monomeric protein of 58.5 kDa. The optimum pH for enzyme activity was about 3.5, and the laccase was most active at 40°C. The N-terminal amino acid sequence of Lcc6 did not correspond to the sequence of Lcc1, which was previously purified from L. edodes. Lcc6 had decolorization activity to some chemical dyes.     

Cloning and Characterization of the Xyn11A Gene from <Emphasis Type="Italic">Lentinula edodes</Emphasis>

Hemicellulose represents a rich source of biomass that can be converted into useful chemical feedstocks. One of the main components of hemicellulose is xylan, a polymer of xylose residues. Xylanase enzymes that hydrolyze xylan are therefore of great commercial interest. We have cloned a gene (xyn11A) that encodes a 283-amino acid xylanase enzyme from the fungus Lentinula edodes. The enzyme has a pI of 4.6 and belongs to the highly conserved glycosyl hydrolase family 11. The xylanase gene was cloned into a Pichia pastoris expression vector that secretes active enzyme into both solid and liquid media. The optimal reaction conditions were at pH 4.5 and 50°C. The enzyme had a K_m of 1.5 mg/ml and a V_max of 2.1 mmol/min/mg. Xyn11A produced primarily xylobiose, xylotriose, and xylotetraose from a birchwood xylan substrate. This is the first report on the cloning of a hemicellulase gene from L. edodes.     

Effect of the physical properties ofLentinula edodes bedlogs on fruiting body production

Fruiting ofLentinula edodes was induced by soaking the bedlogs in water. It appeared that each bedlog had its own optimum water content for fruiting, and the value was affected by the extent of wood decay. A high content of free water, more than 20%, together with a high content of air volume, 32–43%, resulted in good fruiting. As less decayed young bedlogs have high contents of wood substance, it is difficult to obtain high contents of both free water and air volume. This is one reason why less decayed bedlogs produce less fruiting. Contribution No. 323 from the Tottori Mycological Institute.     

Effect of freezing and thawing on cell membranes of Lentinus edodes,the shiitake mushroom

When cell membranes of Lentinus edodes mycelium were rapidly frozen at either 50 or 160°C/min, viability was lost and this correlated with rupture of the plasmalemma and residual membrane material and with alterations in the organelles. Although with slow cooling (1°C/min) 80% of the samples recovered viability, some cells still showed similar changes to those cooled rapidly, indicating that individual cells of the mycelium do not respond in the same way.     

Relationship between the Molecular Structure of the Nitrogen Source and the Activity of the Extracellular Lectins of Lentinus edodes (Berk.) Sing [Lentinula edodes (Berk.) Pegler] upon Submerged Cultivation

The activity of the extracellular lectins of Lentinus edodes (Berk.) Sing [Lentinula edodes (Berk.) Pegler] and the formation of a pigmented mycelial film by this fungus upon submerged cultivation in a synthetic medium were found to depend on the presence of some amino acids (particularly, asparagine) and Ca²⁺ and Mn²⁺ ions in the medium. Quantum-chemical calculations suggest that the different character of the interaction of amino acids with the aforementioned ions is due to differences in the hydrophobicity of the amino acids rather than to differences in the electron structure of the amino acid zwitterions.     

Use of spent mushroom substrates from Agaricus subrufescens (syn. A. blazei, A. brasiliensis) and Lentinula edodes productions in the enrichment of a soil-based potting media for lettuce (Lactuca sativa) cultivation: Growth promotion and soil bioremediation

This study aimed to assess physicochemical and microbiological properties of fresh spent mushroom substrates (SMSs) – without post-crop heat treatment – from Agaricus subrufescens and Lentinula edodes production to optimize the use of these residues in the soil enrichment for lettuce growth promotion and soil remediation. Organic matter and C content of both SMSs were high. Fresh A. subrufescens SMS was a good source of N, P and K. On the other hand, L. edodes SMS presented a lower concentration of these nutrients and a high level of immaturity. Both SMSs presented high electric conductivity values (2.5–3.4 mS/cm). Microbiological analysis, based upon enumeration of culturable bacteria (thermophilic and mesophilic) and fungi, and also evolution of CO₂, showed that SMSs played higher microbial diversity than soil control. Laccase activity from A. subrufescens SMS tended to remain constant during a 2-month period, while L. edodes SMS presented low laccase activity throughout the same period. Agaricus subrufescens and L. edodes were able to grow on a PDA (Potato Dextrose Agar) media supplemented with different concentrations of atrazine (1–50 μg/ml), degraded the herbicide, attaining rates of 35% and 26%, respectively. On experiments of lettuce growth promotion using a soil-based potting media with different SMS rates, 5% and 10% (dw) rates of A. subrufescens SMS resulted in higher lettuce aerial dry weights than the rates of 25% and 40%, the chemical fertilization (NPK) and the control (soil). At 10% supplementation, lettuce aerial dry weight increased 2.2 and 1.3 times compared to the control and the NPK treatment, respectively. Protein content increased along with SMS rates. Fresh A. subrufescens SMS was an excellent supplement for lettuce growth promotion and showed potential for remediation of biocides possibly due to improved microbial diversity and enzymatic activity. Fresh L. edodes SMS was not a good fertilizer, at least under the conditions tested. However, microbiological analysis showed that promising results may be achieved when using fresh L. edodes SMS for soil remediation.     

香菇单孢杂交及耐高温杂交子的ISSR分析

以香菇菌株L135和P18N44为亲本进行单孢杂交,通过显微镜镜检锁状联合以及与亲本的拮抗试验初步获得123个杂交子。以47 ℃热激3 h后菌丝在PDA培养基中的恢复生长情况进行初筛,菌丝在木屑培养基中经历33 ℃高温后的恢复长速进行复筛,共筛选获得耐高温杂交子12个。选用8条引物对12个耐高温杂交子和2个亲本进行了ISSR标记分析,结果表明这12株耐高温杂交子与亲本都存在一定的遗传差异,说明与亲本相比杂交子在基因水平上发生了不同程度的重组,是新的香菇耐高温菌株。