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Open Access
Atypical laccases from the white-rot fungus Pleurotus ostreatus and their application for the treatment of industrial coloured effluents
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Festa, Giovanna; Giardina, Paola; Faraco, Vicenza; Piscitelli, Alessandra; Sannia, Giovanni
White-rot fungi are the most efficient decomposers of lignocellulose because of their capability to synthesize the relevant hydrolytic (cellulases and hemicellulases) and oxidative (laccases, lignin-peroxidases and Mn-peroxidases) extracellular enzymes required to degrade the major components of substrates (cellulose, hemicellulose, and lignin) into low-molecular-weight compounds that can be assimilated in fungal nutrition [1]. Recently, extensive research on these fungi has been conducted with the aim of isolating new organisms able to secrete new enzymes with capability to be used in industrial applications, such as bioremediation of polluted soils and industrial waste-waters, biobleaching and biopulping in pulp and paper industries, textile and food industries, etc. Fungal laccases (benzenediol: oxygen oxidoreductases; EC1.10.3.2) are ligninolytic enzymes that have been isolated from various fungi [2]. They belong to the class of the blue oxidases containing 4 copper atoms/molecule distributed in three different copper binding sites [3, 4]. The type-1 site is responsible for the intense blue colour of the enzyme due to a maximum absorbance at 605 nm; the type-2 site does not exhibit signals in the visible absorbance spectrum; and the type-3 site incorporates two copper centres and is responsible for a band near 330 nm. All these copper ions are involved in the catalytic mechanism. Laccases reduce oxygen to water and simultaneously perform a one electron oxidation of aromatic substrates (polyphenols, methoxysubstituted monophenols, aromatic amines, etc.). These enzymes are present in multiple isoforms, depending on the fungal species and environmental growth conditions [5, 6].
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
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
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
Overexpression of laccases in Coprinopsis cinerea
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Kilaru, S.; Rühl, M.; Saathoff, A.; Dwivedi, R.C.; Zomorrodi, M.; Lange, K.; Majcherczyk, A.; Hoegger, P.J.; Kües, Ursula
Laccases are versatile redox-enzymes that oxidize various phenolic compounds and aromatic amines. Because of the broad substrate specificity, these enzymes are of interest for many different biotechnological applications. White-rot fungi are the major source of laccases. Although basidiomycete species give rise to relatively high enzyme yields, these might not be optimal for applications. Basidiomycetous laccases have been reported to show hyper- or hypo-glycosylation when expressed in ascomycetes. In consequence, enzyme characteristics were found to be altered. Therefore, we use the basidiomycete Coprinopsis cinerea as an organism for laccase overproduction. We present a vector system for easy and rapid cloning of promoters and/or genes of interest and show that such constructs can be functional in laccase production.
Open Access
VI Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI)
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena
This volume summarizes the scientific communications presented at the 6th Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI) held in Pamplona (Spain) from June 3rd to 6th, 2005. GCBB-VI continues the tradition of putting together scientist working with basidiomycetes around the world. Our interest, as organizers, was to strength the communication between groups working on basic and applied research both in the field of edible mushrooms and in that of other industrial applications of these microorganisms. The scientific program included sessions focused on genetics and breeding coordinated by Lucy Ramírez and Rick Kerrigan; Genome Analysis chaired by Allen Gathman; Cellular and Molecular Biology, coordinated by Regina Kahmann and Erika Kothe, Industrial Applications moderated by Giovanni Sannia and Kerry Burton; Plant and Animal Pathogens coordinated by José Pérez-Martín; and Biodiversity coordinated by Philippe Callac. A complete version of the scientific program can be found at the end en of this Volume. In the coffee talks the need of a more active community of scientist working on basidiomycetes was a recurrent topic. After this meeting took place, several proposals for the complete sequencing of basidiomycete genomes are going to be presented for evaluation by groups participating in GCBB-VI.
Open Access
Agaricus devoniensis complex comprises a group of heterothallic isolates constituting a basis for breeding
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Callac, P.; Spataro, C.; Lataillade, E.; Blasi, P.; Guinberteau, J.
A recent phylogenetic reconstruction of Agaricus section Duploannulati revealed that A. devoniensis and A. subfloccosus are two complexes of species close to A. bisporus. The A. subfloccosus complex comprises two homothallic entities, while the A. devoniensis complex was never studied until now. A sample of 26 isolates, some being unreliably determined, were examined to (i) confirm their identity using a PCR-RFLP marker revealing a characteristic A. devoniensis ITS polymorphism, and (ii) for their ability to fruit in standard conditions used for A. bisporus cultivation. Twenty one isolates were confirmed as A. devoniensis, and only two collections from USA were unable to fruit. The five remaining isolates were excluded from the complex and were unable to fruit; their ITS1+2 regions were sequenced and alignments indicated that four of them were similar to A. campestris and that one belonged to a new entity close to A. bitorquis and A. cappellianus. For the 19 fructifying isolates of the complex, we attempted intrastock and interstock mating tests with single spore isolates: for three isolates, we did not get spore germination; and for seven isolates, we observed partial to complete intersterility between strains. The nine remaining isolates exhibited a unifactorial system of sexual incompatibility for which eight different mating type alleles were detected. Within this group, the heterothallic and presumably interfertile isolates differed in their origin (Greece, France), their habitat (dune, coniferous trees), and their morphology (mean spore length: 5.6 to 6.6 μm); they constitute a diversified genetic basis usable to select smooth white and attractive cultivars for this tasteful edible and cultivable species.
Open Access
Selection of Pleurotus ostreatus strains in a genetic breeding program
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Idareta Olagüe, Eneko; Jurado Cabanillas, Javier; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena
The basidiomycete Pleurotus ostreatus, commonly known as oyster mushroom, is the second largest edible mushroom crop behind the white button mushroom, Agaricus bisporus. It accounts for nearly one-quarter of the total worldwide mushroom production. Furthermore, P. ostreatus has a high industrial interest because it is a good source of enzymes and other products with biotechnological, industrial and medical applications, it is easy to cultivate and because of its good organoleptic characteristics. Since of 2003, our group research has carried out genetic breeding programs based on the determination of QTLs controlling production and quality in industrial cultures of this fungus. In this breeding program the first test consisted in putting under fructification conditions 130 strains obtained from the crossing of protoclon PC21 (P. ostreatus var. ostreatus wild strain) by a collection of monokarions derived from N001 (P. ostreatus var. florida commercial strain). For this purpose, 2 kg (3 repetitions per strain) bags of industrial sustrate were inoculated and cultivated at 21ºC. Mature fruiting bodies were collected and weighted daily during the fructification period. The second test was made using the six strains that performed the better in Test1, but were cultivated at 18ºC and with 15 repetitions per strain were performed. From this test, three strains were selected and used in Test3. In this test, other three strains obtained from the crossing between monokarions descending of N001 and selectioned for their high growth rate were introduced. In this test the weight of the bags was increased to 5 kg and the cultures were cultivated at 18ºC. The strains obtained from PC21 have good charactericts for mushroom size, with similar behaviour for yield and precocity. The strains obtained from the crosses between N001 descendants have better mushroom size and similar yield and precocity than N001, then breeding was obtained. The candidate strains for next tests are PC21xMA046 and PC21xMA027 for their high yield and the mushroom good features.
Open Access
Genetic breeding of edible mushrooms: from the genome to the production of new varieties of Pleurotus ostreatus
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Pisabarro de Lucas, Gerardo; Peñas Parrila, María Manuela; Pérez Garrido, María Gumersinda; Park, Sang-Kyu; Eizmendi Goikoetxea, María Arantzazu; Parada Albarracín, Julián Andrés; Palma Dovis, Leopoldo; Idareta Olagüe, Eneko; Jurado Cabanillas, Javier; Castellón Gadea, Jordi; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena
The breeding of new varieties of industrially cultivated edible mushrooms must proceed in the framework defined by the breeding objectives, the biological characteristics of the material and the legal and cultural constraints imposed to the breeding technology to be used. This last aspect is of the greatest importance in the case of a food that is considered in European countries as high quality and closer to nature than other industrially produced foods. This fact prevents the use of genetic-engineering based technologies for breeding, as the consumers would hardly accept genetically modified mushrooms. Consequently, mushroom breeding should be based on time-consuming processes of classic breeding. Molecular biology, however, can offer to the breeders useful tools for speeding up the selection process, for evaluating the new bred lines and, last but not least, to identify and eventually protect legally the outcome of their breeding programs.
Open Access
Ras module function is involved in regulation of sexual development Schizophyllum commune
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Schubert, D.; Kothe, E.
The white rot fungus Schizophyllum commune is used as a model to investigate sexual development in hymenomycetes. We isolated the gene gap1 encoding a GTPase-activating protein for Ras. Disruption of gap1 should therefore lead to strains accumulating Ras in its activated, GTP-bound state and to constitutive Ras signaling. Mating behavior was not altered in Δgap1 monokaryons whereas growth rate in Δgap1 monokaryons was reduced about 25%. Dikaryotic Δgap1/Δgap1 strains displayed 50% growth reduction. Hyphal growth was disturbed showing a wavy growth pattern. In dikaryons, clamp formation was severly disturbed as hook cells failed to fuse with the penultimate cell at the site that in wildtype cells is marked by a peg formed from the mother cell. Instead, the dikaryotic character of the hyphae was rescued by fusion of the hooks with nearby developing branches. The mating type genes of the B factors encoding a pheromone receptor system are known to be required for clamp cell fusion. A role for Ras in the same process in discussed. Fruitbody formation was observed in homozygous Δgap1/Δgap1 dikaryons which, however, formed increased numbers of fruit body primordia, whereas the amount of fruit bodies was not raised. Mature fruit bodies formed no or abnormal gills. No production of spores could be observed. Similar phenotypes in fruitbody development had been previously described for elevated intracellular cAMP levels. Thus, the signalling of Ras is discussed with respect to cAMP signalling.
Open Access
Genetic analysis of Coprinopsis cinerea mutants with defects in fruiting body development
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Srivilai, Prayook; Chaiseana, Wassana; Kües, Ursula
Few genes have so far been cloned and characterized in fruiting of the heterothallic mushroom Coprinopsis cinerea. Fruiting body development normally occurs on the dikaryon. However, the binucleate state of the mycelium hinders easy access of genes. Self-compatible mutants with defects in the mating type pathways can form fruiting bodies without prior fusion to another strain. Uninucleate haploid oidia of such mutants can easily be mutagenized and germinated mycelia tested for defects in fruiting. Mutants can be produced from oidia by classical techniques such as UV treatment or by modern REMI (restriction enzyme-mediated integration) mutagenesis via transformation. Such mutants of self-compatible strains have now been successfully appointed in cloning genes acting in sexual development. Co-isogenic strains of compatible mating types support in genetic characterisation of the mutants.
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
Development of a sporeless strain of oyster mushroom (Pleurotus ostreatus)
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Baars, Johan; Sonnenberg, Anton S.M.; Hendrickx, Patrick
The enormous amounts of spores produced by oyster mushroom (Pleurotus ostreatus) cause lung-related health problems among employees working in oyster mushroom cultivation. If sporeless varieties are used for large-scale cultivation, these lung problems can be avoided. For development of a commercially attractive strain of sporeless oyster mushroom, strain ATCC 58937, a sporeless strain of oyster mushroom was used as a donor of the trait. Microscopic analysis of basidia showed that meiosis was aborted at an early stage. Both nuclear types that constitute strain ATCC 58937 could be retrieved by protoplasting. Protoplasting commercial strain HK35 yielded only one of its nuclear types. Crosses between the ATCC nuclear types and the HK35 nuclear types (either directly or using the Buller phenomenon) yielded normal sporulating strains, indicating that sporelessness was caused by a recessive trait. Among the offspring of crosses between the ATCC nuclear types and the HK35 nuclear types the sporeless trait segregated in a 1 to 1 ratio. The sporeless trait could be mapped and strongly linked genomic markers were developed. The breeding strategy was to successfully introduce sporelessness into both nuclear types of a commercial variety, to achieve a sporeless variety. In a first cross between a sporeless culture and a commercial strain, not only sporelessness was transferred to the commercial variety. Therefore, repeatedly backcrossing the progeny of the first cross with the commercial variety is used to try to restore the original genetic material from the commercial strain as much as possible. Performance of a number of “prototypes” of a sporeless oyster mushroom was tested on commercial mushrooms farms and proved to be satisfactory.
Open Access
In situ RNA-RNA hybridization: a useful method for analysis of the distribution of transcripts of various genes in Lentinula edodes fruiting bodies
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Shishido, Kazuo
The in situ RNA-RNA hybridization was renewed understanding that it is a useful method for analysis of the distribution of transcripts of various genes in fruiting bodies of Lentinula edodes. By using this method, we obtained the following results. Large amounts of the transcripts of ribonucleotide reductase small subunit gene (Le.rnr2) and UMP-CMP kinase gene (uck1), which is a target of developmental regulator PRIB, are present in both hymenium and outer region of trama in the hymenophore (gill tissue). The hymenium is the part for production of basidiospores and the outer region of trama is the region branching out into subhymenium (on the top of which hymenium is formed). The Le.ras transcript is present mostly in outer region of trama and in trama cells, while the transcript of trimeric G-protein αsubunit gene (Le.ga1) is mostly in hymenium. The transcript of mfbC gene, which is the target of PRIB and probably encodes the protein interacting with a putative translation initiation factor 5A (eIF5A), is present in outer region of trama. The transcript of hyd1 (hydrophobin 1 gene), whose expressed product is considered to be involved in the formation in the extracellular matrix of lined air channels with a hydrophobic membrane, is present everywhere in the mycelial tissues of developing fruiting bodies except for the top parts of pileus (cap) and for prehymenophore. The region surrounding prehymenophore contains a high level of the transcript. These results suggest that Le.rnr2 and uck1 genes play a role mainly in the nucleotide biosynthesis essential for production of basidiospores and for divergence of trama cells into subhymenium cells. The Le.ras and mfbC play a role in divergence of mycelial cells and the Le.ga1 plays a role in spore-production. The hydrophobin-mediated air channels may be formed almost all the parts of developing fruiting bodies.
Open Access
QTL mapping of pathogenicity in Heterobasidion annosum sensu lato
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Olson, Ake; Lind, Mårten; Stenlid, Jan
Heterobasidion annosum (Fr.) Bref. sensu lato, casual agent of annosum root rot in conifers, is the economical most devastating forest pathogen in the northern hemisphere. A genetic linkage map of H. annosum, was constructed from a compatible mating between isolates from the North American S and P intersterility groups. The linkage analysis of 358 AFLP markers in 102 progeny isolates generated 19 linkage groups containing 6 or more markers that covered 1468 cM. Two distinct methods were used to analyse the segregation of pathogenicity. 1 year old pine seedlings grown in the greenhouse were infected with H. annosum progeny isolates. Pathogenicity was measured as mean necrosis lengths caused in 10 pine plants. One QTL for pathogenicity was found on linkage group 15 with a LOD peak of 2.95, spanning a 31.2 cM large area. This QTL explained 14.3% of the variation. Another QTL were found in a small linkage group containing 5 markers and spanning 36.8 cM, with a LOD peak of 4.40 at marker paacts02. 19.1% of the variation could be explained by this QTL. The heritability of pathogenicity on pine was estimated to be 0.21 in this study. The disease increase rate values from an in vitro test were used as another estimate of pathogenicity and used for the QTL-analysis. From the in vitro pathogenicity test, a large area of 26.5 cM on linkage group 11 between the markers acgcs5 and paacgp08 contains a high QTL probability of LOD 3.09. This QTL explained 16.4% of the total variation in this experiment. The heritability of pathogenicity on pine was estimated to be 0.088 in this study. Successfully localization of the two intersterility genes (S and P) on the map were carried out through mating of the progeny isolates with three tester strains carrying known intersterility genotypes.
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
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
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
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
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)).