Congresos 2011 y anteriores

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Open Access
Copper in fruiting body development of Coprinus cinereus
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Navarro González, Mónica; Kilaru, S.; Majcherczyk, A.; Kües, Ursula
The model homobasidiomycete Coprinopsis cinerea grows best at 37°C, but, normally, it produces fruiting bodies only at moderate temperatures around 25-28°C. Light is needed to induce fruiting and also for fruiting body maturation. Cultures kept after fruiting induction predominantly in the dark form structures with an extended stipe and an underdeveloped cap (so-called “etiolated stipes”). In a day/night rhythm, caps develop further, basidia are formed, in which karyogamy and meiosis occurs and of which the basidiospores bud off. Besides light, fruiting body development in basidiomycetes has been repeatedly linked to enzymes belonging to the group of phenoloxidases, in particular the multi-copper containing laccases. However, their roles in fruiting remain unclear. In attempts to induce laccase production in liquid standing cultures at 37°C, to our surprise we found unusual inititation of fruiting body development. However, the abundantly formed primordia did never develop into mature fruiting bodies but into large-sized etiolated stipes, both in dark and in light. Laccase under these conditions was not detected in the medium but bound to the fruiting initiating mycelium. Moreover, enzyme production and etiolated stipe formation correlated with an increase from pH 5.5 to a slightly alkaline pH. Ammonium was found to be produced and nitrate reductase activity has enzymatically been shown. Under normal fruiting conditions, addition of copper to cultures enhances fruiting initiation in time and number. To further unravel the potential involvement of laccases in fruiting as well as of proteins influencing ammonia secretion, we are studying expression of corresponding genes during vegetative growth and fruiting body development. Work in our laboratory is supported by DBU (Deutsche Bundesstiftung Umwelt). MNG holds a CONACYT (Mexico) PhD studentship.
Open Access
Sequence analysis and expression of a RecQ gene homologue from Lentinula edodes
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Katsukawa, Shiho; Shishido, Kazuo
We cloned and sequenced a recQ gene homologue from Lentinula edodes. This gene, named Le.recQ, was found to have a coding capacity of 945 amino acids (aa). The deduced Le.RECQ protein was clearly smaller than other fungal RecQ proteins such as Neurospora crassa QDE3 (1955 aa), Schizosaccharomyces pombe Rqh1 (1328 aa), and Saccharomyces cerevisiae SGS1 (1447 aa). It exhibited the highest homology to the Arabidopsis thaliana RecQl4A protein (1182 aa) in its size and aa sequence. Northern-blot analysis showed that the Le.recQ gene is transcribed at similar levels during mycelial development in L. edodes fruiting-body formation. The L. edodes dikaryotic mycelial cells were found to contain a clearly larger amount of Le.recQ transcript than the L. edodes two compatible monokaryotic mycelial cells. Results in situ RNA-RNA hybridization showed that subhymenium and outer region of trama contain larger amounts of Le.recQ transcript. Expression of Le.recQ cDNA in S. cerevisiae might partially complement defects associated with the loss of its homologue S. cerevisiae SGS1 gene.
Open Access
Fungal diversity adds value to biotechnology and agriculture
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Zervakis, Georgios I.
Mediterranean countries host rich biological diversity (genetic, population, species, habitats, communities, ecosystems). Until recently research on the fungal diversity was focusing relatively more on phytopathogenic fungi, invertebrate parasites, and saprotrophic and ectomycorrhizal mushrooms (Pezizales, higher Basidiomycetes). For higher Basidiomycetes in particular, detailed inventories and check-lists have been compiled in many western European countries. In the Mediterranean region, however, pertinent data are limited and fragmentary; only recently new information has started to accumulate. Indicative is the case of Greece, where selected ecosystems are studied in respect to their macromycetes diversity, revealing the existence of taxa with significant ecological and economic interest. Prerequisites for the exploitation of biological resources (incl. fungi) is the availability of a large number of individuals with a wide genetic basis, which are correctly identified and suitably evaluated. For example, elucidating taxonomy and clarifying phylogenetic relationships among Pleurotus species has contributed significantly to their widespread use. Large-scale applications related directly (or indirectly) with mushroom resources and their exploitation include the edible mushroom industry, production of medicinal and health-promoting factors, improvement of soil fertility, remediation of soils, enhanced plant growth, suppressiveness of soil-borne pathogens of plants, animal feed, transformation of xenobiotics and antibiotics, biosorption of toxic elements, decolorization of organic pollutants, degradation of industrial and agroforestry wastes, etc. Particular emphasis is given to the upgrade of lignocellulosic wastes and residues through their detoxification and biotransformation into value-added products; among them, soil conditioners and fertilizers generated from spent mushroom substrates conform with the much-sought notion of sustainability in agriculture.
Open Access
Formation of hyphal loops in xylotrophic coprinoid mushrooms
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Badalyan, Suzanna M.; Kües, Ursula
Recent molecular analysis split the traditional genus Coprinus (Homobasidiomycetes) into four distinct genera: Coprinus, Coprinopsis, Coprinellus and Parasola. Coprinoid mushrooms are usually saprotrophic on soil and/or dung of herbivores. However, more than 60 species are able to grow on wood and straw. Xylotrophic mushrooms are forcing a relatively short supply of nitrogen and phosphorous nutrients. Coprinus comatus has been reported to produce specialized structures (“spiny balls”) to penetrate nematodes for nutrient supply (Luo et al. 2004, Mycologia 96, 1218-1224). Nematode traps of other fungi involve adhesive hyphal network and knobs, hyphal loops and snares. Toxin production may support in nematode immobilisation. Nematode-trapping species belong mainly to the mitosporic Deute romy - ce tes, but some are also found amongst Zygomycetes and Basidiomycetes. We have observed hyphal loops in several wood-decaying basidiomycetes, such as Daedalea quercina, Ganoderma lucidum, Lentinula edodes, Piptoporus betulinus and Pleurotus ostreatus. Furthermore, regular and irregular hyphal loops and/or rings were observed in the four clades of Coprinoid species (Coprinus comatus, Coprinellus angulatus, C. bisporus, C. curtus, C. domesticus, C. disseminatus, C. ellissi, C. micaceus, C. xanthothrix, Coprinopsis cinerea, C. gonophylla, C. radians, C. strossmayeri, C. scobicola, and P. plicatilis). Hyphal loops were particularly often formed in Coprinellus species. Such structures were rare in Coprinopsis atramentaria, C. cothurnata, C. romagnesiana, C. psychromorbida and Coprinus patouillardii (an unclassified isolate). It is not clear yet why Basidiomycetes fungi have these structures. Is it that many species have nematode trapping abilities by formation of such structures? Thanks to the DAAD, NATO and the Deutsche Bundesstiftung Umwelt for financial support.
Open Access
Identification and functional characterisation of ctr1, a Pleurotus ostreatus gene coding for a copper transporter
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Peñas Parrila, María Manuela; Azparren Larraya, María Goretti; Domínguez, A.; Sommer, H.; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza Ekoizpena
Copper homeostasis is primordial for life maintenance and especially relevant for ligning-degrading fungi whose phenol-oxidase enzymes depend on this micronutrient for their activity. In this paper we report the identification of a gene (ctr1), coding for a copper transporter in the white rot fungus Pleurotus ostreatus, in a cDNA library constructed from four-days old vegetative mycelium growing in submerged culture. The results presented here indicate that: (1) ctr1 functionally complements the respiratory deficiency of a yeast mutant defective in copper transport supporting the transport activity of the Ctr1 protein; (2) ctr1 transcription is detected in all P. ostreatus developmental stages (with exception of lamellae) and is negatively regulated by the presence of copper in the culture media; (3) ctr1 is a single copy gene that maps to P. ostreatus linkage group III; and (4) the regulatory sequence elements found in the promoter of ctr1 agree with those found in other copper related genes described in other systems. These results provide the first description of a copper transporter in this white rot fungus and open the possibility of further studies on copper metabolism in higher basidiomyetes.
Open Access
Molecular characterization of A cellobiohydrolase gene family in the fungus Pleurotus ostreatus
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Eizmendi Goicoechea, Arantza; Sannia, Giovanni; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza Ekoizpena
Cellulose is the most abundant biological polymer on Earth. Its chemical composition consists of D-glucose units linked by β-1,4- glycosidic bonds forming linear polymeric chains with a reducing and a non-reducing end. Cellulose chains may either adhere to each other, via hydrophobic and van der Waals interactions, forming crystalline structures or remain more loosely packaged (amorphous cellulose). Consequently, the physical structure and morphology of native cellulose is complex and not uniform. Biological degradation of cellulose depends on the action of three types of enzymes: endoglucanases (E.C.3.2.1.4), cellobiohydrolases (E.C.3.2.1.91) and β-glucosidases (E.C.3.2.1.21). All them hydrolyse β-1,4-glycosidic bonds but they differ on the substrate specificity. Endoglucanases hydrolyse the amorphous regions of the cellulose fibbers generating new reducing and non-reducing ends, cellobiohydrolases attack the molecule ends yielding cellobiose units, and β-glucosidases hydrolyse cellobiose molecules yielding glucose. Cellobiohydrolases can be classified into two groups: type I (CBHI) and type II (CBHII), each having opposite chain-end specificities. CBHI prefer the reducing ends while CBHII act at non-reducing ends. By the screening of a genomic library from the basidiomycete Pleurotus ostreatus var. florida, we have isolated five cbhI genes, named cbhI1, cbhI2, cbhI3, cbhI4 and cbhI5, proving the occurrence of a multigenic family coding for this enzymatic activity. Using this sequences as probe, it has been possible to know the conditions in which are expressed those genes. This has allowed the synthesis of the each gene cDNA and, by comparison of this sequence with the corresponding genomic sequence, the characterization of their structure. On the other hand, using the RFLP technique and a progeny of 80 monokaryons derived from the dikaryon N001, the five genes have been mapped on the linkage map of P. ostreatus var. florida mapping the cbhI1 to the chromosome IV and the others to the chromosome VI.
Open Access
Isolation, molecular characterization and location of telomeric sequences of the basidiomycete Pleurotus ostreatus var. florida
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Pérez Garrido, María Gumersinda; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena
The white rot fungus Pleurotus ostreatus is an edible basidiomycete of increasing biotechnological interest due to its ability to degrade both wood and chemicals related to lignin degradation products. Telomeres are specialized structures at the end of all eukaryotic chromosomes. Ensure chromosome stability and protect the ends from degradation and from fusing with other chromosomes. Telomeres sequences are extraordinary highly conserved in evolution. The loss of telomeric repeats triggers replicative senescence in cells. For identification of restriction telomeric fragments in a previously described linkage map of Pleurotus ostreatus var. florida (Larraya et al., 2000), dikaryotic and eighty monokaryotic genomic DNAs were digested with diferents restriction enzymes (BamHI, BglII, HindIII, EcoRI, PstI, SalI, XbaI and XhoI) electrophoresed and transferred to nylon membranes. Numerous polymorphic bands were observed when membranes were hibridized with human telomericd probe (TTAGGG)132 (heterologous probe). Telomeric restriction fragments were genetically mapped to a previously described linkage map of Pleurotus ostreatus var.florida, using RFLPs identified by a human telomeric probe (tandemly repeating TTAGGG hexanucleotide). Segregation of each telomeric restriction fragment was recorded as the presence vs. absence of a hibridizing band. Segregation data for seventy three telomeric restriction fragments was used as an input table to be analysed as described by Ritter et al. (1990) and by Ritter and Salamini (1996) by using the MAPRF program software. Seventeen out of twenty two telomeres were identified. Telomere and telomere-associated (TA) DNA sequences of the basidiomycete Pleurotus ostreatus were isolated by using a modified version of single- specific-primer polymerase chain reaction (SSP-PCR) technique (Sohapal et al., 2000). Telomeres of Pleurotus ostreatus contain at least twenty five copies of non-coding tandemly repeated sequence (TTAGGG).
Open Access
Natural and artificial hybridization of Agaricus subrufescens Peck (= A. Blazei Murrill sensu Heinemann): lessons from the quasi-alleles of the rDNA ITS1+2 region
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Kerrigan, R.
Agaricus subrufescens Peck was described from both wild and cultivated specimens in 1893. It has been sporadically cultivated in various countries since that time, and is presently an economically important “nutriceutical” food. It is known by several names, including A. rufotegulis Nauta, A. brasiliensis Wasser et al., and A. blazei Murrill sensu Heinemann. A long-term study of diverse isolates and specimens, emphasizing cultural studies and analysis of rDNA ITS1+2 sequences, strongly indicates that a single phylogenetic entity exists. Some interpopulational interfertility has also been demonstrated. Yet the picture is not simple. The species is amphithallic, with complementary reproductive routes, producing recombinant spores with cryptic karyotic states and some self-fertility. Sequences from the Americas were always highly heteromorphic, while those from Hawaii and the UK were homomorphic. This implies that American isolates may be hybrids between (at least) two formerly isolated populations. To test that idea, ITS1+2 sequences from isolate SBS1, an SSI from a California strain, were amplified, cloned and sequenced. Both allelism and recombination are evident in these 711-713 nt sequences: 4 (3+1) parental and 11 recombinant sequences were recovered. The mechanism of fine-scale recombination is unknown (PCR artifacts have not been ruled out). Recombination events exceeded 1.0 per 700 nt. Physical linkage was apparent among 11 polymorphic characters distributed along the ITS1+2. On this basis the parental allelic sequences were deduced, and a comparison with the homomorphic UK sequence was made. The evidence suggests that a European-like strain may have contributed one ITS1+2 allele to an ancestor of the isolate from California. However, if true, “crossovers” must then occurred prior to the origin of the SBS1 SSI, possibly in the SBRF progenitor (or its progenitor(s)).
Open Access
Fungal laccase: properties and aplications
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Jönsson, Leif J.
Laccase (EC 1.10.3.2; benzenediol:oxygen oxidoreductase) was first discovered at the end of the 19th century in the sap of Oriental lacquer trees. Later on, the laccase from the white-rot basidiomycete Trametes versicolor was thoroughly characterized using biochemical and biophysical methods. It is an extracellular blue multicopper glycoprotein. The copper ions are involved in the catalytic process, in which a reducing substrate, typically a phenol, is oxidized and molecular oxygen is reduced to water. Today, a multitude of different laccases and laccase genes from various sources have been characterized. The enzyme seems to have different physiological roles in different types of organisms. Several of the best characterized laccases come from basidiomycete fungi causing white-rot decay of wood. These laccases are generally regarded to be associated with the biodegradation of lignin, although more research is needed to shed light on the fundamental molecular mechanisms. Recent advances with regard to the structural and functional diversity of laccases will be discussed in relation to efforts to clarify the physiological roles of the enzyme and to elucidate its potential in various applications, including detoxification, bleaching, and analysis.
Open Access
Verticillium fungicola cell wall glucogactomannan-binding of the lectin from the Pleurotus ostreatus fruit bodies
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Bernardo, D.; Pérez Cabo, A.; García Mendoza, C.
The Verticillium fungicola mycoparasitism on Agaricus bisporus fruit bodies appears to be a complex process made up of successive steps in which the recognition and binding between complementary molecules, the A. bisporus fruit body lectin and the V. fungicola cell wall glucogalactomannan, have recently been demonstrated. P. ostreatus fruit bodies have been described as containing a lectin and also presenting the “dry bubble” or the Verticillium disease. The aim of the present work is to purify and characterize the P. ostreatus lectin and compare the properties of both lectins in an attempt to confirm if the specific glucogalactomannan-lectin recognition and binding is the necessary step for the V. fungicola mycoparasitism process in P. ostreatus. The characteristics and properties of the purified P. ostreatus lectin together with those also previously described by us on A. bisporus lectin show that, although both lectins present different chemical structures, they behave very similarly in relation to their glucogalactomannan-binding, thus confirming the existence of the specific recognition and binding step in the Verticillium disease on P. ostreatus fruit bodies.
Open Access
Characterisation of Agaricus bisporus response genes to Verticillium fungicola infection
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Costa, A.; Thomas, D.J.I.; Bailey, A.; Foster, G.D.; Challen, M.P.; Mills, P.R.
The mycoparasite Verticillium fungicola is a persistent threat to the cultivation of the mushroom Agaricus bisporus. Mushroom “dry bubble” is characterised by an undifferentiated mass of cells and can result in major crop losses. During the establishment of “dry bubble” substantial changes occur in the biochemistry and physiology of both partners. To enable new insights to be made into the molecular events underlying the disease, work is in progress to identify genes expressed during pathogen infection. Subtractive Suppressive Hybridisation (SSH) has enabled recovery of 65 expressed sequenced tags (ESTs) differentially expressed during infection. After database searches 27 of the genes were identified as most likely from V. fungicola, 25 from A. bisporus and 13 unknown. Bioinformatic analysis suggested that the response genes identified were involved in a range of biological functions that included stress, signalling, protein synthesis and cell wall structure and function. Specific full-length genes will be recovered using cDNA library constructed from lesions of A. bisporus infected with V. fungicola, enabling silencing approaches to be used to further investigate the role of the identified genes in disease. An alternative higher-throughput method of gene function analysis, RNA interference (RNAi) using A. bisporus model genes (URA3, CBX), is also being developed. Silencing constructs expressing RNAi hairpin were transformed into A. bisporus using Agrobacterium tumefaciens and hygromycin resistance. Screening of the transformants by PCR confirmed integration of the silencing construct in 24 transformants. RT-PCR is being used to confirm transcription of the RNAi hairpin. Quantitative PCR will be used to analyse levels of target gene transcripts post RNAi transformation. The role of A. bisporus genes identified, in the infection process, will be determined through infection trails with A. bisporus silenced lines.
Open Access
Study of two acidic proteinases, probably involved in the dimorphism and pathogenicity of Ustilago maydis, basidiomycete of the corn smut disease
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Noriega Reyes, M.Y.; Miramón Martínez, P.; Mercado Flores, Y.; Ramírez Zavala, B.; Hernández Rodríguez, C.; Villa Tanaca, L.
Ustilago maydis is a dimorphic phythopathogenic fungus and the causal agent of the corn smut disease. In this work, the purification and biochemical characterization of the acid proteinases pumAe (extracellular) and pumAi (intracellular) of U. maydis were performed. Also, identity of the gene that encodes for pumAi (PRAum) was explored in the genome of the fungus. The proteases were purified and biochemically characterized. The molecular masses of pumAe and pumAi were 72 and 35.3 kDa respectively. The optimal pH of activity of proteinases was 4.0. The pumAe Km value was of 3.5 μM and a Vmax of 11430 μmol h-1 mg-1 when Suc-R-P-F-H-L-L-V-Y-MCA was used as substrate. The protease pumAi was inhibited by pepstatine A. Yeast-tomycelium transition was inhibited by Pepstatine A in the culture medium. The hypothetical gene that encodes for protease pumAi (PRAum gene) was located in the U. maydis genome project and was amplified by PCR and cloned into TOPO-TA 2.1 plasmid and pNMT-1, a Schizosaccharomyces pombe expression vector. In the U. maydis genome one copy of the gene by Southern blot analyses was detected. In brief, the expression of this gene (PRAum), performed by RT-PCR assays, was regulated by the source of nitrogen. The heterologous expression experiments in S. pombe allowed a fast purification and confirmed that pumAi enzymatic activity was encoded by PRAum gene.
Open Access
Molecular analysis of two acidic proteinases pumAe and pumAi and aminopeptidase pumAPE from Ustilago maydis: enzymes purification and differential expression
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Noriega Reyes, M.Y.; Miramón Martínez, P.; Mercado Flores, Y.; Ramírez Zavala, B.; Hernández Rodríguez, C.; Villa Tanaca, L.
Proteolytic system of Ustilago maydis was recently partially described (Mercado-Flores et al., 2003). Two acidic proteinases pumAe (extracellular) and pumAi (intracellular) and aminopeptidase pumAPE were detected and purified from the haploid phase of U. maydis. Purification consisted of ammonium sulphate fractionation and different chromatographic steps. Molecular masses were estimated: 58 kDa for pumAPE, 72 kDa for pumAe and 35.3 kDa for pumAi. Enzymatic activity was optimal at pH 7.0 and 35 ºC for pumAPE and 4.0 for the two proteinases. pumAPE was inhibited by EDTA-Na2, 1,10-phenanthroline, bestantin, PMSF and several divalent cations, while proteinase pumAi was inhibited by pepstatine A, also finding that yeast-to-mycelium transition was inhibited by Pepstatine A in the culture medium. Primers were designed in order to amplify the gene APEum encoding pumAPE and PRAum gene encoding pumAi, and they were used as probes in a Southern blot. One copy of each gene was detected by genome in several strains. Differential expression of APEum was assessed under different physiological conditions, detecting high expression levels on media supplemented with corn infusion, proline, peptone and ammonium sulphate. PRAum is expressed when cells are exposed to corn infusion and ammonium sulphate.
Open Access
Enzymatic characterization of a monokaryon population of the edible mushroom, Pleurotus ostreatus with a view to genetic improvement
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Terrón, María del Carmen; López, María; Carbajo, José M.; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; González Aldo, E.; Producción Agraria; Nekazaritza Ekoizpena
In this work the lignocellulolytic enzymes produced by the edible mushroom Pleurotus ostreatus var. florida were studied. The objective was to know their relationship with the degradation of the biopolymers present in the cell wall of wheat-straw for the purpose of explaining their influence on the production and quality characters of the fruiting bodies. The following enzymatic activities were studied both in solid and submerged culture: Ligninases (Lignin Peroxidase, Manganese Peroxidase (MnP) and Laccase), Cellulases (Glucohydrolases, Glucosidases) and Hemicellulases from the group Arabinofuran- Xylanases (Xylanase, Xilosidase, Glucoronidase, Arabinofuran-Oxidase and Acetylesterase), cooperating enzymes (Glyoxal Oxidase) and feedback enzymes (Glucose Oxidase (GOD), Aryl Alcohol Oxidase (AAO), Tyrosinase (TYR), Veratryl Alcohol Oxydase (VAO), Cellobiose Dehydrogenase (CDH)). The first studies regarding all the mentioned enzymes were performed using the dikayon (N001) and the parental monokarion strains “fast” (PC9) and “slow” (PC15). The studies on all this whole group of enzymes, which are enough representative of the lignocellulolytic complex, let to conclude that (both in solid or submerged culture) the enzymes of major influence in colonizing the natural substrate and also those whose activity-determination better guarantees their further mapping were Laccases, MnP, AAO and TYR. Subsequently these four activities were measured in the monokaryon population being Laccases and MnP, those yielding the best levels in medium-7 (rich in nitrogen). In addition both enzymes allow the discrimination between “fast-” or “slow-” monokaryon strains both in solid medium with several dyes, or in liquid culture in agitation. The analysis of the enzymatic activities detected in the assayed conditions, in the population of “fast” or “slow” strains let to the observation that they map in different places where the loci corresponding to Laccase (pox) and mnp genes are located. These results open the possibility to design more precise studies that could help to establish a correlation between the contribution of the genes already described and the activity of the different ligninolytic enzymes. In addition the results will contribute to know whether in P. ostreatus genome there are new genes or if they correspond with locations that regulate these enzymatic activities, or it is a gene that has a role in the transport system or a kind of effector in the exportation machinery of the protein to the culture medium.
Open Access
Monstruosities under the inkcap mushrooms
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Navarro González, Mónica; Domingo Martínez, A.; Navarro González, S. S.; Beutelmann, P.; Kües, Ursula
Four different Inkcaps were isolated from horse dung and tested for growth on different medium. In addition to normal-shaped mushrooms, three of the isolates formed fruiting body-like structures resembling the anamorphs of Rhacophyllus lilaceus, a species originally believed to be asexual. Teleomorphs of this species were later found and are known as Coprinus clastophyllus, respectively Coprinopsis clastophylla. The fourth of our isolates also forms mushrooms but most of them are of crippled shape. Well-shaped umbrella-like mushrooms assigns this Inkcap to the clade Coprinellus. ITS sequencing confirmed that the first three strains and the Rhacophyllus type strain belong to the genus Coprinopsis and that the fourth isolate belongs to the genus Coprinellus.
Open Access
Anticancer activity of polysaccharides produced by Pleurotus ostreatus in submerged culture
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Daba, A.; El Demellawy, M.; El Eshasy, H.
It has been known for many years that some compounds produced by edible mushrooms encompass anticancer activities. Most of production methods were based on cultivation of mushroom in solid medium. In the present study Pleurotus ostreatus mycelia were grown in submerged culture. The cultivation of fungal cells in submerged culture resulted in higher growth rate with better control of production process. The bioactive polysaccharides (both intracellular and extracellular) were extracted from culture by solvent repeated precipitation. The polysaccharide structure was determined by examining NMR, IR spectra and the primary structure of the polysaccharide was mainly glucan. The 13C NMR spectral pattern indicated the polysaccharides are highly branched with mainly 1→3 and 1→6 linkage. The results of in vitro anti cancer studies demonstrate that this type of polysaccharides possesses anticancer activity against human oesophageal cancer cell line. Moreover, in the course of in vitro studies, mushroom polysaccharides showed anti-tumour activity and also considered to be biological response modifier because of their mechanism of action through stimulation of the immune system. The polysaccharide activity is especially beneficial in clinics when used as an adjuvant with chemotherapy to decrease its side effect. This work describes production process of anti cancer compound( s) by mushrooms and suitable for pharmaceutical industries.
Open Access
Expression profiling of natural antisense transcripts in Agaricus bisporus
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Eastwood, D.C.; Sergeant, M.; Mead, A.; Burton, K.S.
Our view of the role of RNA has changed from being a passive intermediary of genetic information to acting as a regulator of gene expression in the form of short-interfering RNA, micro RNA, riboswitches and natural antisense. Long length natural antisense transcripts (NATs) have been identified for genes which are up-regulated after harvest in the fruitbody of the mushroom Agaricus bisporus. These NATs therefore are likely to be involved in the regulation of postharvest events such as development and senescence. A novel quantitative reverse transcriptase PCR technique has been developed. The data have been statistically analysed to produce expression profiles of NATs for six postharvest genes. The average antisense/sense ratios varied by three orders of magnitude, from 8.0 for shs13 to 6 x 10-3 for cruciform DNA binding protein. The expression profiles were found to be highly specific for individual genes, to be dynamic over time and highly variable between neighbouring tissues. This latter characteristic has led to the speculation that NATs may be involved in tissue differentiation. Sequence information of natural antisense transcripts form A. bisporus suggests that they are synthesized from messenger (sense) RNA by RNA-dependent RNA-polymerase. Evidence will be presented that to support the hypothesis that the level of antisense may be controlled by the 3’ processing of sense RNA.
Open Access
Wild strains of Agaricus bisporus: a source of tolerance to dry bubble disease
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Largeteau, M.L.; Baars, Johan; Juárez del Carmen, S.; Regnault Roger, C.; Savoie, J.M.
The button mushroom Agaricus bisporus is susceptible to various pests and diseases. Dry bubble, caused by the Hyphomycete Verticillium fungicola, is currently the more serious disease and is worldwide in distribution. Cultivars are susceptible and the pathogen develops resistance towards the very few fungicides admitted at mushroom farms. Breeding for resistance is necessary and wild strains of A. bisporus are putative sources of tolerance to V. fungicola.We present results on the susceptibility of some wild strains of the INRA-CTC collection and the PPO MRU collection. Besides the severity of the disease, the strains were also compared for their ability to develop each of the symptoms induced by the pathogen: spotted mushrooms, stipe blow-out and spheroid masses (the bubbles) which are the typical symptom of the disease. Agaricus bisporus 2100, cultivated in numerous French mushroom farms, was used to assess the aggressiveness of various isolates of V. fungicola var. fungicola, the variety responsible for the disease in Europe at present. Significant variability in aggressiveness was observed. Isolate VCTC, which induced severe symptoms on A. bisporus 2100 (30-40% of diseased mushrooms), revealed interesting tolerance (10-18% of diseased mushrooms) among five wild A. bisporus strains and hybrids between wild strains. A cross test was performed with two cultivars and seven wild stains of A. bisporus contaminated with five V. fungicola isolates, two of var. fungicola and three of var. aleophilum, the latter identified as responsible for the disease in USA and Canada. The wild strains screened in this experiment were far more tolerant than the cultivars, exhibiting 3-9% of diseased mushrooms compared to 20-22%. All the strains were more susceptible to the pathogens of var. aleophilum than to those of var. fungicola. These experiments showed that very tolerant material exists in collection and can be used as parents to breed for resistance. The greater susceptibility of A. bisporus to V. fungicola var. aleophilum must be taken into consideration in breeding programmes, this variety being present in North America and being isolated in Europe in the past.
Open Access
Mapping the Pleurotus ostreatus genome
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Castellón Gadea, Jordi; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena
Pleurotus ostreatus is a commercially important edible mushroom commonly known as oyster mushroom which has also important biotechnical applications. Industrial production of P.ostreatus is based on a solid fermentation process in which a limited number of selected strains are used. Optimization of industrial mushroom production depends on improving the culture process and breeding new strains with higher yields and productivities. In a previous study a linkage map of P. ostreatus strain N001 was constructed, which provided a basis for performing an efficient QTL (Quantitative trait loci) analysis based in a population of 80 sibling monokaryons. The map is based on the segregation of RAPD markers, RFLP markers, phenotypic characters and cloned genes. Nevertheless the linkage map is just a first step towards the selection of the appropiate parentals for new breeds. In order to organize and improve the access to the data and information accumulated in the previous works mentioned above, a Microsoft® Excel Linkage Map Matrix (MELMM) was designed and created. On this linkage map matrix we could have an easy and functional view of the P. ostreatus linkage map data, such as, recombination frequencies, genotypes information and degree of similarity between monokaryons that will help us in the design of breeding crosses aimed at improving QTLs of agronomic interest of new commercial strains.
Open Access
Nutritional value of protein from vegetative mycelia of edible mushroom Pleurotus ostreatus
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Parada Albarracín, Julián Andrés; Urdaneta, Elena; Marzo Pérez, Florencio; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza Ekoizpena; Ciencias del Medio Natural; Natura Ingurunearen Zientziak
The present work was designed to study the effects of supplementation a control diet with P. ostreatus mycelium for evaluation a nutritional value of mycoprotein and possible cholesterol lowering.