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
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
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
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
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
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
Heterologous expression of mating type genes in basidiomycetes
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Srivilai, Prayook; James, Timothy Y.; Vilgalys, Rytas; Chaiseana, Wassana; Kües, Ursula
Mating type genes in basidiomycetes encode two types of transcription factors (HD1 and HD2) and pheromones and pheromone receptors. Usually, mating type genes are so dissimilar in DNA sequence (allelic genes and genes from different species) that they do not cross-hybridize. In homobasidiomycetes, directly next to the A mating type locus encoding the transcription factors is a highly conserved gene mip that allows positional cloning. A candidate gene for positional cloning of the B mating type genes encoding the pheromone-pheromone receptor system is cla4/ste20. With more and more mating type loci cloned from different species, evolution of these loci and their genes can be addressed by sequence analysis and by function by transformation into other species, here Coprinopsis cinerea. Transformation of cloned mating type genes into heterologous hosts can lead to activation of mating type controlled development. Heterologous expression of mating type genes is especially interesting for species in which no transformation system exists. Since in C. cinerea an A null-mutant is available without functional transcription factor genes, self-compatibility of cloned A genes from homothallic species can also be tested.
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
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
Virus related symptoms in crops of the button mushroom Agaricus bisporus
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Sonnenberg, Anton S.M.; Lavrijssen, Brian
Since more than five years symptoms are seen in Dutch mushroom crops that correlate with the presence of a set of dsRNAs. These dsRNAs are indicative for the presence of viruses. Symptoms are discoloration of white mushrooms varying from cream to brown. Crops can be heavily affected with more than 50% of the mushrooms showing discoloration. Mostly, however, crops show mild symptoms with a few percentages of mushrooms showing an off-white color. Since traiders associate this discoloration with low quality economical damage can be substantial for individual farmers. In affected crops up to 15 dsRNAs are observed with varying intensity. Only the five shortest dsRNAs show a perfect quantitative and qualitative correlation with the symptom. They are always present in discolored mushrooms and the intensity of the bands seen in agarose gels vary with the intensity of the discoloration. Most of the other dsRNAs are also present in affected crops but are also seen in crops without any visual symptoms. We have started a molecular analysis of the dsRNAs in order to design sensitive tests for each dsRNA. This will reveal if all or some dsRNAs are always present in mushrooms or are derived from an unknown infectious source. From a number of dsRNAs 60 to 80% of the sequence has been determined. RT-PCR tests have been designed recently for these dsRNAs and the first result of tests of several crops will be presented.
Open Access
Rat cytochrome P450-mediated transformation of dichlorodibenzo-Pdioxins by recombinant white-rot basidiomycete Coriolus hirsutus
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Shishido, Kazuo
Rat cytochrome P450, CYP1A1, has been reported to play an important role in the metabolism of mono-trichlorodibenzo-p-dioxins (M-TriCDDs). To breed lignin (and M-TetraCDDs)-degrading basidiomycete Coriolus hirsutus strains producing rat CYP1A1, an expression cassette [C. hirsutus gpd promoter-C. hirsutus gpd 5’-portion (224-bp of 1st exon-8th base of 4th exon)-rat cyp1a1 cDNALentinula edodes priA terminator] was constructed and inserted into pUCR1 carrying the C. hirsutus arg1 gene. The resulting recombinant plasmid, MIp5-(cyp1a1+arg1) was introduced into protoplasts of C. hirsutus monokaryotic strain OJ1078 (Arg-, Leu-), obtaining three good Arg+ transformants. These transformants [ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), and ChTF5-6(CYP1A1)] were estimated to carry nine, six, and seven copies of the expression cassette on their chromosomes, respectively. Immunoblot analysis revealed that the three transformants produce similar amounts of rat CYP1A1 enzyme. ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), ChTF5-6(CYP1A1) and recipient OJ1078 were cultivated in a liquid medium containing 2,7/2,8 (at a ratio of 1:1)-dichlorodibenzo-pdioxins (2,7/2,8-DCDDs) and the amount of intra- and extracellular 2,7/2,8-DCDDs remaining was measured. The results showed that all three transformants efficiently transform 2,7/2,8-DCDDs through the action of the recombinant rat CYP1A1 enzyme.
Open Access
Computational prediction of protein-coding gene and annotation of DNA sequences with agronomic interest in Pleurotus ostreatus (Oyster Mushroom)
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Palma Dovis, Leopoldo; Peñas Parrila, María Manuela; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza Ekoizpena
Pleurotus ostreatus, commonly known as oyster mushroom, is a commercially important edible fungus with interesting biotechnological properties. Quantitative trait loci (QTL) analyses are rare in fungi and little is known about their number, position, and genetic structure. Previous studies of our group have allowed the construction of a genetic linkage map of P. ostreatus var. florida, which has provided the basis for performing an efficient QTL analysis. In fact, there is a region of the chromosome VII of P. ostreatus where the most QTLs related to the production and precocity characters have been mapped. These quantitative traits are presumably under the control of a polygenic genetic system and could be associated with some chromosomal regions. The hypothesis of this work is that there is a region in the chromosome VII of protoclon PC15 (monokaryotic parental of the N001 dikaryotic strain) where exist genes which are responsible for the QTLs mentioned above. In order to test this hypothesis, we are developing a molecular QTL analysis through the sequencing of a region with an approximated size of 320 Kbp in chromosome VII (protoclon PC15). For this purpose, a BAC genomic library was constructed and two BAC clones spanning the region of interest are being sequenced. To carry out an efficient computational prediction of protein-coding genes and its annotation on the partial sequences obtained up to date, we have used different Internet resources such as BLASTx, BLASTp, BLASTn, and FGENESH trained on some basidiomycetes genomic data like Phanerochaete chrysosporium and Cryptococcus neoformans (SoftBerry). To our knowledge, this is the firs molecular QTL analysis performed on this edible mushroom.
Open Access
The roles of SC3 and SC15 of Schizophyllum commune during growth on wood
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Jong, Jan F. de; Smant, Wiecher; Boer, Wietse de; Wösten, Han A. B.; Lugones, Luis G.
The SC3 hydrophobin plays several roles in growth and development of S. commune. It lowers the surface tension of the aqueous substrate, enabling aerial growth, and it coats the aerial hyphae rendering them hydrophobic. SC3 also allows hyphae to attach to hydrophobic surfaces. Moreover, SC3 has a role in the cell wall architecture. In the absence of the hydrophobin, S. commune produces more mucilage. The SC15 protein mediates aerial hyphae formation and attachment in the absence of SC3. Besides being secreted into the medium, the protein can be found in the mucilage that binds aerial hyphae together. So far, studies were performed on minimal media. We here assessed the roles of SC3 and SC15 during growth on wood. SC3 and SC15 were shown not to play a role in colonization of wood. Biomass formation and radial extension was similar in the wild-type and in strains in which either or both SC3 and SC15 were deleted. Interestingly, in contrast to growth on minimal medium deletion of SC15 alone affected formation of aerial hyphae. A similar reduction was observed for the SC3 mutant while reduction in the ∅SC3∅SC15 was most dramatic. At the moment we assess the role of SC15 in the fruiting body. Preliminary data using GFP as a reporter indicate that SC15 is expressed within the fruiting body. Here it may play a role in the mucilage surrounding the hyphae.
Open Access
Species identification and detection of fungi in biological materials by FTIR microscopy
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Naumann, Anette; Navarro González, Mónica; Peddireddi, Sudhakar; Schützendübel, Andres; Kües, Ursula; Polle, Andrea
FTIR spectroscopy provides the opportunity to simultaneously detect many molecular bonds or functional groups of different polysaccharides, proteins, lipids, aromatic and other compounds. The measurement principle is based on the absorption of infrared light by dipolar molecular bonds. In combination with microscopy, local resolution of the chemical composition is possible. Each absorption point or peak in the spectrum can be integrated to create an image of the distribution of the corresponding compound. We use FTIR-microscopy in order to detect fungi in plant tissues such as in infected wood and in mycorhizal roots. For the development of a fast and inexpensive method for localisation and identification of fungi, differences between FTIR measurements of fungi and plant cells are characterized. In addition, FTIR spectra of different fungi are compared. Beech wood blocks were infected with Trametes versicolor and with Schizophyllum commune and FTIR spectra in sections of the infected wood determined. Cluster analysis revealed major differences between FTIR spectra recorded from wood fibres and empty vessel lumina and spectra from fungal mycelium, irrespectively of whether grown on the surface of wood or inside vessel lumina. Species specific clustering of spectra of fungal mycelium grown on the wood surface and inside vessel lumina demonstrated the potential of FTIR microscopy to identify fungal mycelium in wood. Currently, we are sampling FTIR spectra from various basidiomycetes in order to define species according to their specific FTIR spectra. The work is supported in frame of the Lower Saxony Competence Network for Sustainable Timber Utilisation (NHN) by the Ministry of Culture of Lower Saxony and EFRE. The group of UK is funded by the DBU. MNG holds a PhD studentship from CONACYT, Mexico.
Open Access
Multiple hydrophobin genes in mushrooms
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Peddireddi, Sudhakar; Velagapudi, R.; Hoegger, P.J.; Majcherczyk, A.; Naumann, Anette; Olbrich, A.; Polle, Andrea; Kües, Ursula
Hydrophobins are small secreted fungal proteins that form amphipathic films on the hyphal surfaces. In the wood-rotting fungus Schizophyllum commune, four different hydrophobins are known with well established functions during vegetative growth and fruiting body development. Our study aims at elucidating the role of these proteins in wood penetration and lignocellulose degradation. Blast searches of the genome of the dung fungus Coprinopsis cinerea revealed a surprising number of 34 different hydrophobin genes in this species. Functional analysis of these genes is in progress.
Open Access
Wild strains of Agaricus bisporus: a source of tolerance to dry bubble disease: resumen de póster
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Largeteau, M.L.; Baars, Johan; Regnault Roger, C.; Savoie, J.M.
Agaricus bisporus is susceptible to various pests and diseases. Dry bubble, caused by Verticillium fungicola, is currently the most serious disease and is distributed worldwide. All cultivars are susceptible and the pathogen develops resistance towards the very few fungicides admitted. Breeding for resistance is necessary and wild strains of A. bisporus are putative sources of tolerance. We present results on the susceptibility (severity of the disease, ability to develop the various symptoms) of some wild strains of the INRA-CTC and the PPO MRU collections. A commercial strain revealed significant variability in agressiveness among isolates of V. fungicola var. fungicola responsible for the disease in Europe at present. Isolate VCTC, which induced severe symptoms revealed interesting tolerance 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 isolates, two of var. fungicola and three of var. aleophilum, the latter responsible for the disease in USA and Canada. The wild strains screened were far more tolerant (3-9% of diseased mushrooms) than the cultivars (20-22%). All the strains were more susceptible to the var. aleophilum than to the var. fungicola isolates. 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
Cell wall-associated redox enzymes in white rot fungi
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Dwivedi, R.C.; Zomorrodi, M.; Kües, Ursula; Majcherczyk, A.
Many enzymes of white rot fungi involved in wood degradation belong to the class of redox enzymes. The most important are laccase (copper-containing polyphenol oxidase), lignin peroxidase, manganese- dependent peroxidase and manganese-independent peroxidase. However, the role of these enzymes in wood degradation remains unclear and complex redox processes or unknown redox enzymes also may contribute to this process. Several oxidative enzymes secreted by white rot fungi into the environment have been studied in the past, but little attention has been paid to the cell wall-associated redox enzymes. Cell wall-associated laccase activity in the purified cell walls of copper induced cultures of Trametes versicolor has been found. Laccases have been extracted by establishing new methods for cell wall purification and for protein release from the cell walls of basidiomycetes.
Open Access
Mushroom breeding program in Iran
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Gordan, H.R.; Farsi, M.
Mushroom cultivation is a newly established, but growing industry in Iran. There are about 120 producers with a total of about 20,000 tones per year in the country. Agaricus bisporus consists of more than 85 percents of the total production of all mushrooms produced in Iran. Its yield average is about 12-15 kg/m2, while in the global production it is about 27-30 kg/m2. This is mainly due to using strains of genetically weak performance. Since ten years ago a breeding program was started with emphasis on breeding high yielding strains in Mashhad. The short-term effort consisted of selection among single spore isolates and multispores cultures with a better performance in yield. The long-term effort consisted of employing of heterosis in hybrid strains. To reach the aim, more than 350 homokaryone isolates were selected through RAPD markers followed by yield trials from commercial and domestic strains, and crosses were made in many combinations using diallel method. Selection among spores of commercial strains could somehow recover their potential genetic capacity, so that an average of 22 Kg/m2 was recorded in the selected strains. Using growth type as a marker, it was possible to decrease the number of isolates in final stages of selection for homokaryones in solid medium or spawn. Cluster analysis based on average of band numbers emerged by RAPD markers, could separate homokaryotic and heterokaryotic isolates in two distinct groups. Some hybrids showed a better mycelia growth and a considerable higher yield than their parents. Efforts are now being made to collect wild strains of Agaricus bisporus in Iran and evaluating them for desirable genes including resistance to pest and diseases.
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
Investigations into the fungal-fungal interaction between Verticillium fungicola and Agaricus bisporus
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Collopy, P.D.; Amey, R.; Challen, M.P.; Mills, P.R.; Bailey, A.; Foster, G.D.
Plant and animal pathogens section The cultivated button mushroom, Agaricus bisporus, is amenable to number of pathogenic threats including bacteria, viruses, mites, insects and fungi. Currently, the most significant threat to the commercial mushroom industry is the mycoparasite, Verticillium fungicola. Infection by V. fungicola can drastically reduce the yield and value of mushroom crops. The severity of this disease is dependent on the developmental stage of A. bisporus at the time of infection and is manifested in three types of symptoms: spotty cap, stipe blowout and dry bubble. An aim of our research has been to develop molecular tools for V. fungicola that will allow us to study the interaction between this pathogen and A. bisporus. These tools have included transformation methods, marker gene techniques as well as gene-knockout technologies. This has involved the use of Agrobacterium and T-DNA to introduce disruption constructs into V. fungicola as part of a molecular investigation into this fungal-fungal interaction. We have developed an efficient transformation system for V. fungicola that we have now adapted to give high levels of targeted mutagenesis. This technique has successfully generated targeted mutants of a β-1-6 glucanase homologue from Trichoderma harzianum and a Mitogen Activated Protein Kinase homologue (PMK1) from Magnaporthe grisea identified using degenerate PCR primers. We have also developed TDNA tagging technology in a mycology context for random mutagenesis in V. fungicola.