Impacts and tolerance responses of Coprinus comatus and Pleurotus cornucopiae on cadmium contaminated soil.

Large amounts of cadmium (Cd) have been discharged into soil with the rapid development of industry. In this study, we revealed the impacts of Coprinus comatus (C. comatus) and Pleurotus cornucopiae (P. cornucopiae) on soil and the tolerance responses of macrofungi in the presence of Cd by the analysis of soil biochemical properties and macrofungi growth indexes. Results showed that with the cultivation of C. comatus and P. cornucopiae, the HOAc-extractable Cd in soil individually reduced by 9.53% and 11.35%, the activities of soil urease, acid phosphatase, dehydrogenase, and Fluorescein diacetate (FDA) hydrolysis increased by 18.11-101.45%, 8.39-18.24%, 9.37-55.50% and 28.94-41.92%, respectively. Meanwhile, different soil bacterial communities were observed with various macrofungi cultivations. Also, Cd accumulation significantly enhanced the macrofungi antioxidant enzyme activities, which increased by 24.10-45.43%, 30.11-61.53% and 7.03-26.81% for catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activities in the macrofungi, respectively. Moreover, the enhanced macrofungi endophytic bacterial diversities with Cd existence was firstly observed in the present experiment. These findings revealed the possible Cd resistance mechanisms in macrofungi, suggesting C. comatus and P. cornucopiae were promising ameliorators for Cd contaminated soil.

Optimization of cultivation conditions of fermented shaggy ink cap culinary-medicinal mushroom, Coprinus comatus (O.Mull.:Fr.) Pers. (higher Basidiomycetes) rich in Vanadium.

The present paper is mainly aimed at optimization of cultivation conditions of fermented mushrooms of Coprinus comatus rich in vanadium (CCRV). Initial screening of effects of carbon source, temperature, pH, and inoculum size were done by using a one-factor-at-a-time method. The results obtained in that study showed that the optimal medium composition was 30 g glucose/Lin YEPG medium, initial pH 6.0, inoculum volume 10%, and incubation time 120 h. Then the medium was subjected to screening of the most significant parameters using the L9 orthogonal array to solve multivariable equations simultaneously. The results obtained in this study showed that the optimal medium composition was 0.4% V and 30 g glucose/Lin YEPG medium, initial pH 5.0, inoculum volume 15%, and incubation time 120 h. At this medium composition, the mycelial biomass and V content were 7.18 ± 0.24 g/L and 3786.0 ± 17 µg/g, respectively. The anti-diabetic potential of CCRV produced with the optimal level was tested in alloxan-induced diabetes. After the mice were administered (i.g.) with CCRV, the level of blood sugar in the CCRV group was very close to that of the control group. These findings suggested that CCRV produced with the optimal level is useful in the control of diabetes mellitus.

Xylaria coprinicola, a new species that antagonizes cultivation of Coprinus comatus in China.

The species known in China as the chicken-claw fungus is described as a new species, Xylaria coprinicola. This species is known as an antagonist of cultivation of the edible mushroom Coprinus comatus. Stromata of X. coprinicola are cylindrical, terminate in a sterile apex and arise in fascicles from a relative large submerged base; perithecia are immersed and have conspicuously conical ostiolar openings; ascospores are minute. Phylogenetic analyses based on combined partial sequences of rpb2, ß-tub and α-act genes showed that X. coprinicola is closely related to those Xylaria species exclusively associated with termite nests.

Comparison of the structures and prebiotic-like effects in vitro of polysaccharides from Coprinus comatus fruit body and mycelium.

Many effects of Coprinus comatus are attributed to its polysaccharide components. Therefore, the aim of this article is to take Coprinus comatus polysaccharides as the research topic to estimate the difference between the polysaccharides of Coprinus comatus fruiting bodies (CBPs) and the intracellular polysaccharides of liquid fermentation (ICPs). The total carbohydrate contents, monosaccharide compositions, molecular weights, functional groups, microstructures and functional properties of the two prepared polysaccharides were evaluated. At the same time, the influences of the two polysaccharides on the proliferation of lactobacillus and Bifidobacterium in vitro were compared. The structural analysis exhibited that there were slight differences in the two prepared polysaccharides. However, both ICPs and CBPs could be utilized by these two strains. Furthermore, the effects of the two prepared polysaccharides on the proliferation of the selected probiotics were dose-dependent manners within the scope of the experiment, and the ICPs group and CBPs group had no significant difference (P > 0.05). Therefore, this work demonstrates that ICPs could be an equivalent replacer for CBPs.

DNA polymerase mu interacts with a meiosis-specific RecA homolog Lim15 during meiosis in Coprinus cinereus.

Meiosis is a fundamental process in eukaryotes. Homologous chromosomes are paired and recombined during meiotic prophase I, which results in variation among the gametes. However, the mechanism of recombination between the maternal and paternal chromosome is unknown. In this study, we report on the identification of interaction between Coprinus cinereus DNA polymerase mu (CcPol mu) and CcLim15/Dmc1, a meiosis-specific RecA-like protein, during meiosis. Interaction between these two proteins was confirmed using a GST-pull down assay. A two-hybrid assay revealed that the N-terminus of CcPol mu, which includes the BRCT domain, is responsible for binding the C-terminus of CcLim15. Furthermore, co-immunoprecipitation experiments indicate that these two proteins also interact in the crude extract of the meiotic cell. A significant proportion of CcPol mu and CcLim15 is shown to co-localize in nuclei from the leptotene/zygotene stage to the early pachytene stage during meiotic prophase I. Moreover, CcLim15 enhances polymerase activity of CcPol mu early in the reaction. These results suggest that CcPol mu might be recruited by CcLim15 and elongate the D-loop structure during homologous recombination in meiosis.

Life history and developmental processes in the basidiomycete Coprinus cinereus.

Coprinus cinereus has two main types of mycelia, the asexual monokaryon and the sexual dikaryon, formed by fusion of compatible monokaryons. Syngamy (plasmogamy) and karyogamy are spatially and temporally separated, which is typical for basidiomycetous fungi. This property of the dikaryon enables an easy exchange of nuclear partners in further dikaryotic-monokaryotic and dikaryotic-dikaryotic mycelial fusions. Fruiting bodies normally develop on the dikaryon, and the cytological process of fruiting-body development has been described in its principles. Within the specialized basidia, present within the gills of the fruiting bodies, karyogamy occurs in a synchronized manner. It is directly followed by meiosis and by the production of the meiotic basidiospores. The synchrony of karyogamy and meiosis has made the fungus a classical object to study meiotic cytology and recombination. Several genes involved in these processes have been identified. Both monokaryons and dikaryons can form multicellular resting bodies (sclerotia) and different types of mitotic spores, the small uninucleate aerial oidia, and, within submerged mycelium, the large thick-walled chlamydospores. The decision about whether a structure will be formed is made on the basis of environmental signals (light, temperature, humidity, and nutrients). Of the intrinsic factors that control development, the products of the two mating type loci are most important. Mutant complementation and PCR approaches identified further genes which possibly link the two mating-type pathways with each other and with nutritional regulation, for example with the cAMP signaling pathway. Among genes specifically expressed within the fruiting body are those for two galectins, beta-galactoside binding lectins that probably act in hyphal aggregation. These genes serve as molecular markers to study development in wild-type and mutant strains. The isolation of genes for potential non-DNA methyltransferases, needed for tissue formation within the fruiting body, promises the discovery of new signaling pathways, possibly involving secondary fungal metabolites.

The exp1 gene essential for pileus expansion and autolysis of the inky cap mushroom Coprinopsis cinerea (Coprinus cinereus) encodes an HMG protein.

The homobasidiomycete Coprinopsis cinerea is a member of the fungi known as inky cap mushrooms, and its fruiting-body pileus autolyzes soon after completion of the development. During the last 3h of the development, the pileus exhibits umbrella-like expansion: the pileal tissue is cracked at the base of each gill and then each gill tissue is split to form a V-shape, as seen in a cross section. We identified two C. cinerea mutants defective in both pileus expansion and autolysis. The defects in both mutants are due to recessive mutations in a single gene, designated exp1. The exp1 gene is predicted to encode an HMG1/2-like protein with two HMG domains. The transcription of exp1 is strongly induced in the pileus 3h before pileus expansion. This result, together with the fact that the exp1 mutations cause a specific developmental phenotype, suggest that Exp1 is a novel, transcriptional regulator controlling the final phase of fruiting-body morphogenesis.

Molecular analysis of pcc1, a gene that leads to A-regulated sexual morphogenesis in Coprinus cinereus.

A homokaryotic strain (5337) in our culture stock of Coprinus cinereus produced fertile fruit bodies after prolonged culture. Microscopic examination revealed that hyphae dedifferentiated from the tissues of one of the fruit bodies, as well as all basidiospore derivatives from the fruit body, exhibited pseudoclamps, whereas vegetative hyphae of 5337, from which the fruit body developed, had no clamp connections. Genetic analysis showed that the formation of pseudoclamps results from a recessive mutation in a gene designated pcc1 (pseudoclamp connection formation), which is distinct from the A and B mating type genes. Cloning and sequencing of the pcc1 gene and cDNA identified an ORF of 1683 bp interrupted by one intron. Database searches revealed that pcc1 encodes an SRY-type HMG protein. The HMG box shared 44, 41, and 29% sequence identities (>80 amino acids) to those of FPR1 of Podospora anserina, MAT-Mc of Schizosaccharomyces pombe, and prf1 of Ustilago maydis, respectively. Northern analysis revealed that the level of pcc1 expression is higher in the dikaryon, in homokaryons in which the A and B mating type developmental sequences are individually activated, than in the homokaryon in which these sequences are not active. Sequencing of the pcc1-1 mutant allele revealed that the mutant carries a nonsense mutation at serine 211, a residue located between the HMG box and the C terminus. Based on these results, possible roles of the pcc1 gene in the sexual development of homobasidiomycetes are discussed.

Kinetics of stem gravitropism in Coprinus cinereus: determination of presentation time and "dosage-response" relationships using clinostats.

The sensitivity to gravitational stimulation of excised stems of the mushroom fruit body of Coprinus cinereus was determined using clinostat rotation to remove partially-stimulated stems from the normal unidirectional gravitational field. For the strain and conditions tested, the presentation time (the minimum time of stimulation required to elicit a gravitropic reaction) was determined to be 9.6 min. This is the first time the presentation time has been determined for a fungal gravitropic response. Constructional details are given of the clinostats employed in the research and their further use is discussed.

The role of calcium accumulation and the cytoskeleton in the perception and response of Coprinus cinereus to gravity.

The role of Ca2+ in the gravitropic perception and/or response mechanism of Coprinus cinereus was examined by treating stipes with inhibitors of Ca2+ transport and calmodulin. Inhibitors had no effect on gravity perception but significantly diminished gravitropism. It is concluded that, under the conditions tested, Ca2+ is not involved in gravity perception by Coprinus stipes, but does contribute to transduction of the gravitropic impulse. The results would be consistent with regulation of the gravitropic bending process requiring accumulation of Ca2+ within a membrane-bound compartment. Treatment of stipes with an actin inhibitor caused a significantly delayed response, a result not observed with the Ca2+ inhibitors. This suggests that cytoskeletal elements may be involved directly in perception of gravity by Coprinus stipes while Ca(2+)-mediated signal transduction may be involved in directing growth differentials.

[Degradation of plant waste by Coprinus truncorum using 2 culture methods]. / Degradación de residuos vegetales por Coprinus truncorum usando dos métodos de cultivo.

Degradation of yard wastes by Coprinus truncorum growing in a vertical aereated bioreactor or in flasks was studied. There was a constant decay of reducing sugars in the medium that avoided their accumulation and their possible repression of degradative enzymes. Endoxylanase activity at first showed a similar pattern in both culture conditions, with maximal activity on the 12th day, but flasks maintained a high activity thereafter. Flasks also showed a higher endoglucanase activity with a peak on the 18th day, whereas the maximal value in the bioreactor was reached on the 26th day. No Mn-peroxidase and only low values of laccase activity were found. The measurements of pH and soluble proteins during the incubation period were suitable indicators of the degradation process by C. truncorum.

Decolorization of synthetic dyes by crude and purified laccases from Coprinus comatus grown under different cultures: the role of major isoenzyme in dyes decolorization.

Coprinus comatus laccase isoenzyme induction and its effect on decolorization were investigated. The C/N ratio, together with aromatic compounds and copper, significantly influenced laccase isoenzyme profile and enzyme activity. This fungus produced six laccase isoenzymes in high-nitrogen low-carbon cultures but much less in low-nitrogen high-carbon (LNHC) cultures. The highest laccase level (3.25 IU/ml), equivalent to a 12.6-fold increase compared with unsupplemented controls (0.257 IU/ml), was recorded after 13 days in LNHC cultures supplemented with 2.0 mM 2-toluidine. Decolorization of twelve synthetic dyes belonging to anthraquinone, azo, and triphenylmethane dyes, by crude laccases with different proportion of isoenzymes produced under selected culture conditions, illustrated that the LacA is the key isoenzyme contributed to dyes decolorization especially in the presence of 1-hydroxybenzotriazol, which was further confirmed by dyes decolorization with purified LacA in the same condition. The crude laccase only was able to decolorize over 90 % of Reactive Brilliant Blue K-3R, Reactive Dark Blue KR, and Malachite Green, and higher decolorization for broader spectrum of synthetic dyes was obtained in presence of redox mediator, suggesting that C. comatus had high potential to decolorize various synthetic dyes as well as the recalcitrant azo dyes.

A mutation in the FHA domain of Coprinus cinereus Nbs1 Leads to Spo11-independent meiotic recombination and chromosome segregation.

Nbs1, a core component of the Mre11-Rad50-Nbs1 complex, plays an essential role in the cellular response to DNA double-strand breaks (DSBs) and poorly understood roles in meiosis. We used the basidiomycete Coprinus cinereus to examine the meiotic roles of Nbs1. We identified the C. cinereus nbs1 gene and demonstrated that it corresponds to a complementation group previously known as rad3. One allele, nbs1-2, harbors a point mutation in the Nbs1 FHA domain and has a mild spore viability defect, increased frequency of meiosis I nondisjunction, and an altered crossover distribution. The nbs1-2 strain enters meiosis with increased levels of phosphorylated H2AX, which we hypothesize represent unrepaired DSBs formed during premeiotic replication. In nbs1-2, there is no apparent induction of Spo11-dependent DSBs during prophase. We propose that replication-dependent DSBs, resulting from defective replication fork protection and processing by the Mre11-Rad50-Nbs1 complex, are competent to form meiotic crossovers in C. cinereus, and that these crossovers lead to high levels of faithful chromosome segregation. In addition, although crossover distribution is altered in nbs1-2, the majority of crossovers were found in subtelomeric regions, as in wild-type. Therefore, the location of crossovers in C. cinereus is maintained when DSBs are induced via a Spo11-independent mechanism.

Blue light signaling inactivates the mating type genes-mediated repression of asexual spore production in the higher basidiomycete Coprinopsis cinerea.

Monokaryotic mycelia of several wild-type strains of the homobasidiomycete Coprinopsis cinerea form abundant numbers of oidia both in the light and dark due to the regulation of oidia production by the A and B mating type genes. Nevertheless, little is known about whether and how the mating type loci and light signal regulate the oidiation in C. cinerea. Herein, the experimental results demonstrated that the self-compatible homokaryon AmutBmut strain, the mycelia whose nuclei carry mutations in both the A and B loci, can produce only a few oidia in the dark, whereas the formation of numerous numbers of oidia is induced by the light. The semi-compatible homokaryon AmutB, but not ABmut, has the production and behavior of oidia formation similar to those of AmutBmut. These findings indicated that in AmutBmut strain the mutation at the A locus results in repression of oidiation in the dark and the blue light alleviates this effect, whereas the mutated B genes function has no effects. Since, the oidia production relies on both A and light signal, it is possible that A locus might be linked to the blue light receptor genes. The present results demonstrated for the first time that the secondary hyphal knot formation (skn1), fruiting body maturation (mat) and basidiospore formation (bad) genes which are essential in the C. cinerea fruiting pathway are not involved in the regulation of asexual sporulation. In addition, the positive light effect on oidiation could also occur in C. cinerea dikaryons.

Knockdown of LIM15/DMC1 in the mushroom Coprinus cinereus by double-stranded RNA-mediated gene silencing.

The basidiomycete Coprinus cinereus has many advantages as a model organism for studying sexual development and meiosis, but it has been difficult to investigate using reverse-genetics methods, such as gene disruption by homologous recombination. Here, gene repression by dsRNA-mediated gene silencing was tried as an alternative method for reverse-genetics studies. It was shown that transformation of the LIM15/DMC1 dsRNA expression construct (LIM15dsRNA) resulted in genomic insertion of LIM15dsRNA and paucity of the LIM15/DMC1 transcript. First, LIM15dsRNA was transformed into the homothallic strain AmutBmut to generate a homozygote in which both nuclei had a copy of LIM15dsRNA. The LIM15/DMC1-repressed strain showed abnormal homologous chromosome synapsis during meiosis. Basidiospore production was reduced to 16 % by the induction of dsRNA. However, approximately 60 % of basidiospores were viable. Next, a heterozygote was generated in which one nucleus had a copy of LIM15dsRNA. The phenotype was similar to that of the homozygote. These results are not only the first demonstration of dsRNA-mediated gene silencing in a member of the homobasidiomycete fungi, to which 90 % of mushroom species belong, but also the first successful use of a reverse-genetics approach in C. cinereus research.

Molecular genetics of sexual development in the mushroom Coprinus cinereus.

Sexual development in the mushroom Coprinus cinereus is under the control of two mating type loci, A and B. When two haploid homokaryons with compatible alleles at both A and B loci are mated, the coordinated activities of A- and B-regulated pathways lead to formation of a mycelium termed the dikaryon, in which the two nuclei from the mating partners pair in each cell without fusing. The dikaryon is a prolonged mycelial stage that can be induced to develop a multicellular structure, the mushroom, under proper environmental conditions. The two nuclei fuse in specialized cells on the mushroom and immediately undergo meiosis to complete the sexual life cycle. It has been established recently that the A genes encode two classes of homeodomain proteins while the B genes encode pheromones and their receptors. More recently, molecular genetics has been used to reveal genes that work downstream of the mating type genes to regulate dikaryon formation, mushroom morphogenesis, and meiosis.

The control of meiosis progression in the fungus Coprinus cinereus by light/dark cycles.

Meiosis progression in Coprinus cinereus is controlled by light/dark cycles. Light is essential to propel basidia into karyogamy and light intensity determines the timing of meiotic events. The higher the light intensities, the faster the fruiting bodies enter karyogamy. The critical period when light has this influence is between 16 and 6 h before karyogamy. The control is highly stage specific. A 3-h dark period is essential for a Java dikaryon and the Japanese A(mut)B(mut) homokaryon to enter meiotic metaphase; without it the fruit body is permanently arrested at diffused diplotene. This arrest is light intensity-dependent (>20 hlx) and temperature-dependent (e.g., 27 degrees C). The placement of the dark period is very stage specific; it has no effect when placed before karyogamy stage. A dikaryon of London origin is light blind and able to complete meiosis under continuous high light regime. Fruiting bodies arrested under a continuous high light can be rescued by a 3-h dark treatment, but there is always an 8-h lag time to enter meiotic metaphase. It is possible that the dark effect signals cellular processes leading to division events. Cytological studies of arrested fruiting bodies showed that chromosomes are normal in meiotic prophase through pachytene and diplotene, but are unable to undergo chromosome condensation. Genetic crosses between a monokaryon of Java stock J6;5.4 and a monokaryon BL55 or H5 of London stock showed that light-blindness is dominant, and is controlled by a single Mendelian gene.

White-cap mutants and meiotic apoptosis in the basidiomycete Coprinus cinereus.

Among many white-cap mutants of Coprinus cinereus, four distinct classes have been identified cytologically. Mutants of one class progress through meiosis normally but fail to sporulate; the defect is post-meiotic and it triggers apoptosis in the tetrad stage. Mutants of the other three classes have defects in meiotic prophase and these are: (1) those that assemble synaptonemal complexes (SCs) normally; (2) those that assemble axial elements (AEs) but not SCs; and (3) those that assemble neither AEs nor SCs even though the chromosomes are condensed and also paired. All three meiotic mutant classes arrest at meiotic metaphase I and the arrest triggers meiosis-specific apoptosis showing characteristic chromatin condensation, DNA fragmentation as shown by the TUNEL assay, cytoplasmic shrinkage, and finally total DNA degradation. Apoptosis is very cell-type specific; it occurs only in the basidia while the neighboring somatic cells are perfectly healthy and the mushroom continues to develop and mature with very few basidiospores produced. The meiotic apoptosis in C. cinereus is under strict cell cycle control rather than at any time after defect; apoptosis is triggered only after entry to meiotic metaphase. It is intriguing to note that C. cinereus has two checkpoints for arrest and entry to apoptosis: one is meiotic at the metaphase I spindle checkpoint regardless of the time of defects, and one is post-meiotic at the tetrad stage. This is in striking contrast to multiple checkpoint arrests and entries to meiotic apoptosis found in the mouse.

Distribution of mechanical stress is not involved in regulating stipe gravitropism in Coprinus cinereus.

Removal of large segments of the apical part of the stipe of Coprinus cinereus (extending to about half its length) affected neither the ability of the stipe to show gravitropic bending nor its ability to compensate the curvature so induced and adjust to the vertical. However, gravitropic reaction time was directly proportional to the amount of stipe removed. Application of lateral loads of up to 20 g had no adverse effects on adjustment of the stipe to the vertical and continued vertical growth. It is concluded that sensing the distribution of extracellular mass and/or mechanical stress is unlikely to be a component of the control of gravitropic bending in C. cinereus stipes.