Encapsulation of Ammoides pusila Essential Oil into Mesoporous Silica Particles for the Enhancement of Their Activity against Fusarium avenaceum and Its Enniatins Production.

Essential oils (EOs) that have antifungal activity and mycotoxin reduction ability are candidates to develop bioactive alternatives and environmentally friendly treatment against Fusarium species in cereals. However, their practical use is facing limitations such as high volatility, UV sensitivity, and fast oxidation. Encapsulation techniques are supposed to provide protection to the EOs and control their release into the environment. Ammoides pusilla essential oil (AP-EO) proved to be an efficient inhibitor of Fusarium avenaceum growth and its enniatins (ENNs) production. In the present work, AP-EO was encapsulated, using the impregnation method, into mesoporous silica particles (MSPs) with narrow slit pores (average diameter = 3.1 nm) and coated with chitosan. In contact assays using an agar medium, the antifungal activity of AP-EO at 0.1 µL mL-1 improved by three times when encapsulated into MSPs without chitosan and the ENNs production was significantly inhibited both in coated and non-coated MSPs. Controls of MSPs also inhibited the ENNs production without affecting the mycelial growth. In fumigation experiments assessing the activity of the EO volatile compounds, encapsulation into MSPs improved significantly both the antifungal activity and ENNs inhibition. Moreover, coating with chitosan stopped the release of EO. Thus, encapsulation of an EO into MSPs improving its antifungal and antimycotoxin properties is a promising tool for the formulation of a natural fungicide that could be used in the agriculture or food industry to protect plant or food products from the contamination by toxigenic fungi such as Fusarium sp. and their potential mycotoxins.

Experimental Outcrossing in Agaricus bisporus Revealed a Major and Unexpected Involvement of Airborne Mycelium Fragments.

Agaricus bisporus var. bisporus, the button mushroom, has a predominantly pseudohomothallic life cycle. Most of its spores are heterokaryotic and give rise to fertile heterokaryons. However, previous studies have suggested that outcrossing should not be rare in wild populations. In order to discover how outcrossing occurs, we experimentally favored it between aerial propagules of a fruiting donor mycelium and a delayed receiver mycelium that only invaded culture trays. Two donor/receiver pairs were studied, and potentially hybrid basidiomata collected on the receiver trays were analyzed with a mitochondrial marker, two unlinked nuclear CAPS markers, then haplotype markers based on DNA sequences obtained after PCR cloning of the rDNA ITS region and the fruk gene. For one of the two pairs, most basidiomata were hybrids between the donor and the receiver. Genotyping of the hybrids revealed only two genotypes consistent with outcrossing involving airborne mycelium fragments rather than basidiospores. The resident receiver heterokaryon that provided its mitochondria to the hybrid basidiomata is suspected to have had a trophic contribution to their growth and successful fruiting. The high level of heterozygosity and the cultivar introgression previously revealed in wild populations of this pseudohomothallic species may result from outcrossing involving airborne pieces of mycelium.

Characterization of Fusarium acuminatum: A Potential Enniatins Producer in Tunisian Wheat.

Fusarium Head Blight (FHB), caused by multiple species of Fusarium in small grain cereals, is a significant and long-standing problem anywhere in the world. Knowing regional Fusarium spp. present on non-symptomatic grains and their potential for mycotoxin production is of concern for identifying novel actions for FHB and mycotoxin management, such as treatments with essential oils. Analyzing the mycotoxin content of grains from non-symptomatic ears of different wheat varieties cultivated in Tunisia, we isolated Fusaria specimens identified as F. culmorum and F. acuminatum using analysis of the partial DNA sequence of the ß-tubulin gene and ITS region. Two isolates of the latter species, uncommon in cereal grains in this region until now, were shown to be effective producers of enniatins in vitro, with 1390 and 3089 µg g-1 mycelial biomass (dry) in 11-day-old cultures. The susceptibility of an isolate of F. acuminatum to the fungistatic and antimycotoxin effects of eight essential oils was measured. Essential oils from Ammoides pusilla and Thymus capitatus used at 0.1 µL mL-1 in an agar culture medium, affected the mycelial growth by 55% and 79%, respectively and reduced the accumulation of enniatins per unit of mycelial colony by 26% and 52%, respectively. Finally, F. acuminatum was shown to be a contaminant of wheat grains in Tunisia and it may contribute to the contamination in enniatins. Two essential oils of Tunisian plants could be used for developing a biofungicide limiting both its mycelial growth and its accumulation of mycotoxins in grains.

Ammoides pusilla Essential Oil: A Potent Inhibitor of the Growth of Fusarium avenaceum and Its Enniatin Production.

Enniatins are mycotoxins produced by Fusarium species contaminating cereals and various agricultural commodities. The co-occurrence of these mycotoxins in large quantities with other mycotoxins such as trichothecenes and the possible synergies in toxicity could lead to serious food safety problems. Using the agar dilution method, Ammoides pusilla was selected among eight Tunisian plants for the antifungal potential of its essential oil (EO) on Fusarium avenaceum mycelial growth and its production of enniatins. Two EO batches were produced and analyzed by GC/MS-MS. Their activities were measured using both contact assays and fumigant tests (estimated IC50 were 0.1 µL·mL-1 and 7.6 µL·L-1, respectively). The A. pusilla EOs and their volatiles inhibited the germination of spores and the mycelial growth, showing a fungistatic but not fungicidal activity. The accumulation of enniatins was also significantly reduced (estimated IC50 were 0.05 µL·mL-1 for the contact assays and 4.2 µL·L-1 for the fumigation assays). The most active batch of EO was richer in thymol, the main volatile compound found. Thymol used as fumigant showed a potent fungistatic activity but not a significant antimycotoxigenic activity. Overall, our data demonstrated the bioactivity of A. pusilla EO and its high potential to control F. avenaceum and its enniatins production in agricultural commodities.

Methanolic Extracts from Cultivated Mushrooms Affect the Production of Fumonisins B and Fusaric Acid by Fusarium verticillioides.

The maize pathogen Fusarium verticillioides and their mycotoxins cause damage to plants, animals, and human health. This work aimed to evaluate the effect of crude extracts (CEs) from Agaricus subrufescens, Lentinula edodes, and Pleurotus ostreatus fruiting bodies on in vitro production of biomass and mycotoxins by two strains of F. verticillioides. Stipes and pilei were separated before extraction for A. subrufescens and L. edodes. Comparative metabolomics and dereplication of phenolic compounds were used to analyze all CEs. Mushroom CEs did not significantly inhibit the production of mycelial biomass at concentrations of 2 mg mL⁻1. CEs from A. subrufescens (stipes and pilei) and L. edodes pilei inhibited the production of fumonisins B1 + B2 + B3 by 54% to 80%, whereas CE from P. ostreatus had no effect. In contrast, CE from L. edodes stipes dramatically increased the concentration of fumonisins in culture media. Fusaric acid concentration was decreased in cultures by all CEs except L. edodes stipes. Differences in phenolic composition of the extracts may explain the different effects of the CE treatments on the production of mycotoxins. The opposing activities of stipes and pilei from L. edodes offer an opportunity to search for active compounds to control the mycotoxin production by F. verticillioides.

Evolution of Fusarium tricinctum and Fusarium avenaceum mitochondrial genomes is driven by mobility of introns and of a new type of palindromic microsatellite repeats.

BACKGROUND: Increased contamination of European and Asian wheat and barley crops with "emerging" mycotoxins such as enniatins or beauvericin, produced by Fusarium avenaceum and Fusarium tricinctum, suggest that these phylogenetically close species could be involved in future food-safety crises. RESULTS: The mitochondrial genomes of F. tricinctum strain INRA104 and F. avenaceum strain FaLH27 have been annotated. A comparative analysis was carried out then extended to a set of 25 wild strains. Results show that they constitute two distinct species, easily distinguished by their mitochondrial sequences. The mitochondrial genetic variability is mainly located within the intergenic regions. Marks of variations show they have evolved (i) by Single Nucleotide Polymorphisms (SNPs), (ii) by length variations mediated by insertion/deletion sequences (Indels), and (iii) by length mutations generated by DNA sliding events occurring in mononucleotide (A)n or (T)n microsatellite type sequences arranged in a peculiar palindromic organization. The optionality of these palindromes between both species argues for their mobility. The presence of Indels and SNPs in palindrome neighbouring regions suggests their involvement in these observed variations. Moreover, the intraspecific and interspecific variations in the presence/absence of group I introns suggest a high mobility, resulting from several events of gain and loss during short evolution periods. Phylogenetic analyses of intron orthologous sequences suggest that most introns could have originated from lateral transfers from phylogenetically close or distant species belonging to various Ascomycota genera and even to the Basidiomycota fungal division. CONCLUSIONS: Mitochondrial genome evolution between F. tricinctum and F. avenaceum is mostly driven by two types of mobile genetic elements, implicated in genome polymorphism. The first one is represented by group I introns. Indeed, both genomes harbour optional (inter- or intra-specifically) group I introns, all carrying putatively functional hegs, arguing for a high mobility of these introns during short evolution periods. The gain events were shown to involve, for most of them, lateral transfers between phylogenetically distant species. This study has also revealed a new type of mobile genetic element constituted by a palindromic arrangement of (A) n and (T) n microsatellite sequences whose presence was related to occurrence of SNPs and Indels in the neighbouring regions.

Priming to protect maize from Fusarium verticillioides and its fumonisin accumulation.

BACKGROUND: Systemic infection through the seed is one of the routes used by the mycotoxinogenic pathogen Fusarium verticillioides for colonizing maize plants. The prohibition of the use of most chemical fungicides by the EU has promoted research on plant resistance inducers as an effective and sustainable alternative. Induction of a priming state in maize seeds might affect their susceptibility to contamination and accumulation of fumonisins. This state by application of a natural fertilizer called Chamae on maize seeds, was investigated in two varieties to control the colonization by the fungus and the accumulation of fumonisins B1 , B2 and B3 , germinating seeds, dead plants and yield. RESULTS: After inoculation of F. verticillioides on germinating seeds, the colonization by the fungus and the accumulation of fumonisins were significantly lower in seedlings coming from treated seeds, but a significant number of plants stopped their development by necrosis. In a field trial, the 0.01% (v/v) application dilution showed a lower plant density, although the level of biomass at harvest was not affected. CONCLUSION: The priming state contributed to the control of F. verticillioides development from seed infection and fumonisin accumulation in the early stage of plant growth, without affecting the final crop yield, and could reduce fungicide use and environmental contamination. © 2018 Society of Chemical Industry.

Yeast and bacteria from ensiled high moisture maize grains as potential mitigation agents of fumonisin B1.

BACKGROUND: Fumonisin B1 (FB1 ) is a mycotoxin produced by several Fusarium species and is a very common contaminant of maize-based food and feed throughout the world. The selection and use of FB1 -degrading microorganisms appears as a promising alternative to cope with the problem of toxicity towards humans and livestock. High moisture maize grain silage, which is based on natural maize fermentation, could be an interesting reservoir of such microorganisms. RESULTS: Using an in vitro simulated silage model with FB1 naturally contaminated grains, we demonstrated a significant raw decrease in FB1 during ensiling process ascribed to biodegradation mechanisms. A panel of 98 bacteria and yeasts were isolated from this matrix and selected for their ability to use FB1 as the sole source of C and N. For nine of them, the ability to degrade FB1 in vitro was evidenced. Notably, two bacteria identified as Lactobacillus sp. were highlighted for their efficient FB1 -degrading capacity and production of hydrolysed FB1 as intermediate degradation metabolite. CONCLUSION: Fermentation of high moisture maize grain contaminated with FB1 leads to a significant reduction of the toxin and allows the isolation of FB1 -degrading microorganisms that could further be used as FB1 decontaminating agents. © 2016 Society of Chemical Industry.

The Genetic Linkage Map of the Medicinal Mushroom Agaricus subrufescens Reveals Highly Conserved Macrosynteny with the Congeneric Species Agaricus bisporus.

Comparative linkage mapping can rapidly facilitate the transfer of genetic information from model species to orphan species. This macrosynteny analysis approach has been extensively used in plant species, but few example are available in fungi, and even fewer in mushroom crop species. Among the latter, the Agaricus genus comprises the most cultivable or potentially cultivable species. Agaricus bisporus, the button mushroom, is the model for edible and cultivable mushrooms. We have developed the first genetic linkage map for the basidiomycete A. subrufescens, an emerging mushroom crop known for its therapeutic properties and potential medicinal applications. The map includes 202 markers distributed over 16 linkage groups (LG), and covers a total length of 1701 cM, with an average marker spacing of 8.2 cM. Using 96 homologous loci, we also demonstrated the high level of macrosynteny with the genome of A. bisporus The 13 main LG of A. subrufescens were syntenic to the 13 A. bisporus chromosomes. A disrupted synteny was observed for the three remaining A. subrufescens LG. Electronic mapping of a collection of A. subrufescens expressed sequence tags on A. bisporus genome showed that the homologous loci were evenly spread, with the exception of a few local hot or cold spots of homology. Our results were discussed in the light of Agaricus species evolution process. The map provides a framework for future genetic or genomic studies of the medicinal mushroom A. subrufescens.

Spore behaviors reveal a category of mating-competent infertile heterokaryons in the offspring of the medicinal fungus Agaricus subrufescens.

Strain breeding is much less advanced in the edible and medicinal species Agaricus subrufescens than in Agaricus bisporus, the button mushroom. Both species have a unifactorial system of sexual incompatibility, a mating type locus tightly linked to a centromere, and basidia producing both homokaryotic (n) and heterokaryotic (n + n) spores. In A. bisporus, breeding is mainly based on direct selection among the heterokaryotic offspring and on hybridization between homokaryotic offspring. The parental heterozygosity is highly maintained in the heterokaryotic offspring due to suppression of recombination and preferential pairing in the spores of nuclei, each one per second meiotic divisions; such "non-sister nuclei" heterokaryons are fertile. In A. subrufescens, recent studies revealed that recombination is not suppressed and that nuclei from the same second meiotic division can also be paired in a spore that give rise to a "sister nuclei" heterokaryon in which the nuclei bear the same mating type allele. The objective of the present work was to investigate the potential function of the different categories of spores in A. subrufescens and their possible use in a genetic breeding program. Using eight co-dominant molecular markers, we found that half of the offspring of the A. subrufescens strain WC837 were heterokaryotic, one quarter of them being sister nuclei heterokaryons. These heterokaryons were infertile and behaved like homokaryons, being even able to cross between each other. In contrast, non-sister nuclei heterokaryons could fruit but inconsistently due to inbreeding depression. Potential roles of these two categories of heterokaryons in nature and consequences for strain breeding are discussed.

Selected wild strains of Agaricus bisporus produce high yields of mushrooms at 25 °C / Cepas silvestres seleccionadas de Agaricus bisporus con capacidad para producir un alto rendimiento a temperaturas de 25°C

Background. To cultivate the button mushroom Agaricus bisporus in warm countries or during summer in temperate countries, while saving energy, is a challenge that could be addressed by using the biological diversity of the species. Aims. The objective was to evaluate the yield potential of eight wild strains previously selected in small scale experiments for their ability to produce mature fruiting bodies at 25 °C and above. Methods. Culture units of 8 kg of compost were used. The yield expressed as weight or number per surface unit and earliness of fruiting were recorded during cultivation in climatic rooms at 17, 25 or 30 °C. Results. Only strains of A. bisporus var. burnettii were able to fruit at 30 °C. At 25 °C they produced the highest yields (27 kg m−2) and had best earliness. The yields at 25 °C for the strains of A. bisporus var. bisporus ranged from 12 to 16 kg m−2. The yield ratios 25 °C/17 °C ranged from 0.8 to 1.2. Conclusions. The variety burnettii originated in the Sonoran Desert in California showed adaptation for quickly producing fruiting bodies at high temperature when humidity conditions were favorable. Strains of the variety bisporus showed interesting potentials for their ability to produce mature fruiting bodies at higher temperature than present cultivars and might be used in breeding programs (AU)

Antecedentes. El cultivo del champiñón (Agaricus bisporus) en países de clima tropical, o durante el verano en países de clima templado, además del ahorro energético que supone, es un reto que podría abordarse con el uso adecuado de la diversidad biológica de la especie. Objetivos. El objetivo del presente estudio fue examinar el rendimiento potencial de ocho cepas silvestres, previamente seleccionadas en experimentos a pequeña escala, para determinar su capacidad para producir frutos maduros a temperaturas ≥25 °C. Métodos. Se utilizaron unidades experimentales de cultivo con 8 kg de compost. El rendimiento se expresó como peso o número de frutos producidos por unidad de superficie; en naves climatizadas, a una temperatura de 17, 25 o 30 °C, durante todo el cultivo, se registró el momento de fructificación. Resultados. Sólo las cepas de A. bisporus var. burnettii fueron capaces de fructificar a 30 °C. Éstas produjeron un alto rendimiento a 25 °C (27 kg/m2) y la fructificación aconteció más temprano. El rendimiento a 25 °C para las cepas de A. bisporus var. bisporus varió de 12 a 16 kg/m2, con una proporción 25 °C/17 °C de 0,8 a 1,2. Conclusiones. La variedad burnettii, originaria del desierto de Sonora en California, está adaptada para producir cuerpos fructíferos a temperaturas elevadas cuando las condiciones de humedad son favorables. Para las cepas de la variedad bisporus se demostró un interesante potencial para producir cuerpos fructíferos maduros a una mayor temperatura que la utilizada en los cultivares actuales y puede ser utilizada en los programas de mejora genética (AU)

The intraspecific variability of mitochondrial genes of Agaricus bisporus reveals an extensive group I intron mobility combined with low nucleotide substitution rates.

Intraspecific mitochondrial variability was studied in ten strains of A. bisporus var. bisporus, in a strain representative of A. bisporus var. eurotetrasporus and in a strain of the closely related species Agaricus devoniensis. In A. bisporus, the cox1 gene is the richest in group I introns harboring homing endonuclease genes (heg). This study led to identify group I introns as the main source of cox1 gene polymorphism. Among the studied introns, two groups were distinguished according to the heg they contained. One group harbored heg maintained putatively functional. The other group was composed of eroded heg sequences that appeared to evolve toward their elimination. Low nucleotide substitution rates were found in both types of intronic sequences. This feature was also shared by all types of studied mitochondrial sequences, not only intronic but also genic and intergenic ones, when compared with nuclear sequences. Hence, the intraspecific evolution of A. bisporus mitochondrial genome appears characterized by both an important mobility (presence/absence) of large group I introns and by low nt substitution rates. This stringent conservation of mitochondrial sequences, when compared with their nuclear counterparts, appears irrespective of their apparent functionality and contrasts to what is widely accepted in fungal sequence evolution. This strengthens the usefulness of mtDNA sequences to get clues on intraspecific evolution.

Selected wild strains of Agaricus bisporus produce high yields of mushrooms at 25°C.

BACKGROUND: To cultivate the button mushroom Agaricus bisporus in warm countries or during summer in temperate countries, while saving energy, is a challenge that could be addressed by using the biological diversity of the species. AIMS: The objective was to evaluate the yield potential of eight wild strains previously selected in small scale experiments for their ability to produce mature fruiting bodies at 25°C and above. METHODS: Culture units of 8 kg of compost were used. The yield expressed as weight or number per surface unit and earliness of fruiting were recorded during cultivation in climatic rooms at 17, 25 or 30°C. RESULTS: Only strains of A. bisporus var. burnettii were able to fruit at 30°C. At 25°C they produced the highest yields (27 kg m(-2)) and had best earliness. The yields at 25°C for the strains of A. bisporus var. bisporus ranged from 12 to 16 kg m(-2). The yield ratios 25°C/17°C ranged from 0.8 to 1.2. CONCLUSIONS: The variety burnettii originated in the Sonoran Desert in California showed adaptation for quickly producing fruiting bodies at high temperature when humidity conditions were favorable. Strains of the variety bisporus showed interesting potentials for their ability to produce mature fruiting bodies at higher temperature than present cultivars and might be used in breeding programs.

Screening of biological, morphological, and molecular characteristics of single-spore isolate collections of the straw mushroom, Volvariella volvacea (higher Basidiomycetes) from India.

Of the 2 parent strains and 6 single-spore isolates (SSIs) used in this study, the SSIs BBSR-007 and BBSR-002 of the culinary-medicinal straw mushroom Volvariella volvacea exhibited superior growth characteristics on different growth media. These also took less time until first harvest (days after spawning), gave higher numbers of fruiting bodies per unit weight of substrate, and provided a higher mushroom yield on composted substrate. The fruiting body weight of isolate BBSR-007 was significantly higher compared to BBSR-002. On pasteurized paddy straw, the SSI BBSR-007 had a higher mushroom yield than BBSR-002. The contents of dry matter, protein, and other elements (sodium, potassium, and calcium) were on par in both the parent strains and different SSIs, except SSI OE-55-08, which had highest dry matter, protein content, and potassium/-to-odium ratio. In amplified ribosomal DNA restriction analysis, the SSIs did not show any distinctness, whereas in amplified fragment length polymorphism, the SSI OE-55-08 of parent strain OE-55 formed a separate clade; of 4 SSIs of strain OE-274, the SSI BBSR-003 formed a separate clade than other SSIs. The genetically distinct SSI OE-55-08 produced the highest numbers of fruit bodies per unit weight of substrate and exhibited the highest amounts of dry matter and protein in its fruit bodies. Another slightly distinct SSI, BBSR-003, formed white aerial mycelia on growth substrate, and its fruit bodies exhibited the highest levels of sodium and calcium.

Deciphering the ability of Agaricus bisporus var. burnettii to produce mushrooms at high temperature (25 °C).

The button mushroom Agaricus bisporus is cultivated almost worldwide. Its cultivation is standardized and a temperature of 16-19 °C is needed during the fruiting period. The development of A. bisporus cultivars able to fruit at higher temperature (FHT) represents a promising alternative to reduce energy costs during cultivation in hot countries as well as in temperate countries during the hot season. A. bisporus var. burnettii is able to fruit at 25 °C. Understanding the biological mechanisms that underlie such a thermo-tolerance is a prerequisite to further development of breeding strains. The foundation of the FHT ability of the var. burnettii was dissected using a combination of candidate gene approaches and genetic tools. Transcript profiling of A. bisporus var. burnettii at two developmental stages (primordium P and sporophore SP) under two fruit-producing temperature conditions (17 °C and 25 °C) were established by cDNA-AFLP. The expression patterns were more similar within the same stage at the two different temperatures rather than between stages under the same temperature. Only one transcript-derived fragment (TDF) sequence differentially expressed between temperatures was recovered but it could not be further characterized. Twenty-nine TDF sequences differentially expressed between development stages were obtained. The phenotypic assessment of an intervarietal A. bisporus var. bisporus×A. bisporus var. burnettii progeny demonstrated the complex inheritance of the FHT trait. Two quantitative trait loci (QTL) involved in the number of fruit bodies yielded at 25 °C were found on LG II and LG VI. Two functional candidate genes known to be potentially involved in A. bisporus thermo-tolerance, a heat shock protein (HSP70) gene and a gene coding for a para-aminobenzoic acid synthase (PABA), were found in the vicinity of the QTL on LG II. Several positional candidate genes have been also identified in the confidence interval of the QTL on LG VI and are promising for further fine mapping purpose.

Do spawn storage conditions influence the colonization capacity of a wheat-straw-based substrate by Agaricus subrufescens?

Storage conditions of the spawn of edible fungi are of major importance to facilitate the production of mushrooms. Here, standard storage conditions at 10°C or 15°C were used and the potential of colonization of standard European compost by the tropical species Agaricus subrufescens was assessed during the spawn running phase. Two lignocellulolytic activities, laccase and CMC-cellulase, were enhanced after storage compared to control as well as substrate transformation, as described by the aromaticity ratio and a humification ratio calculated from NMR data. This result indicates that mycelium growth probably occurred during storage at 10 or 15°C, leading to a larger amount of biomass in the inoculum. Moreover, the microbial functional diversity of the substrate was favored, showing that the electivity of the substrate was maintained. Thus, these findings indicate that recommendations for the mushroom producers can be established for A. subrufescens cultivation under European standard conditions.

The first set of expressed sequence tags (EST) from the medicinal mushroom Agaricus subrufescens delivers resource for gene discovery and marker development.

Agaricus subrufescens is one of the most important culinary-medicinal cultivable mushrooms with potentially high-added-value products and extended agronomical valorization. The development of A. subrufescens-related technologies is hampered by, among others, the lack of suitable molecular tools. Thus, this mushroom is considered as a genomic orphan species with a very limited number of available molecular markers or sequences. To fill this gap, this study reports the generation and analysis of the first set of expressed sequence tags (EST) for A. subrufescens. cDNA fragments obtained from young sporophores (SP) and vegetative mycelium in liquid culture (CL) were sequenced using 454 pyrosequencing technology. After assembly process, 4,989 and 5,125 sequences were obtained in SP and CL libraries, respectively. About 87% of the EST had significant similarity with Agaricus bisporus-predicted proteins, and 79% correspond to known proteins. Functional categorization according to Gene Ontology could be assigned to 49% of the sequences. Some gene families potentially involved in bioactive compound biosynthesis could be identified. A total of 232 simple sequence repeats (SSRs) were identified, and a set of 40 EST-SSR polymorphic markers were successfully developed. This EST dataset provides a new resource for gene discovery and molecular marker development. It constitutes a solid basis for further genetic and genomic studies in A. subrufescens.

Characterization of the aap1 gene of Agaricus bisporus, a homolog of the yeast YAP1.

The structure, homologies, polymorphism and expression profiles of a new gene, aap1, have been studied for precisely characterizing it and defining its putative involvement in thermo-tolerance of both vegetative mycelium growth and sporophore differentiation. Sequence polymorphism was analyzed in 3 homokaryons of A. bisporus and 24 strains having different abilities for mycelial growth at temperatures above 30 °C and for producing mature fruiting bodies at 25 °C. The level of gene expression was measured by real-time PCR both in vegetative mycelium after transfer from 25 to 32 °C and in primordia and fruiting bodies produced during cultures at 17 or 25 °C. The results indicated that aap1 gene belong to a new subfamily of the yeast YAP1 homologs. It is not a dominant contributor to the thermo-tolerance of A. bisporus, but the protein it encodes may be involved as an overall stress resistance transcription factor. The way Aap1 senses redox level differs from that of AP-1-like transcription factor Yap1.

Optimization of the cultivation conditions for mushroom production with European wild strains of Agaricus subrufescens and Brazilian cultivars.

BACKGROUND: The almond mushroom Agaricus subrufescens (formerly Agaricus blazei or Agaricus brasiliensis) is cultivated at commercial level in Brazil and some Asian countries on local substrates and casing mixtures. Despite its tropical origin, A. subrufescens might be a seasonal option for mushroom growers in western countries, where some wild strains have been isolated. For this purpose, cultivation conditions were developed starting from the substrate and casing mixture commonly used for commercial production of the button mushroom Agaricus bisporus in France. RESULTS: The commercial compost, based on wheat straw and horse manure, used for A. bisporus and the casing mixture (peat and limestone) supplemented with fine sand proved efficient to grow A. subrufescens. Increasing the depth of the casing layer improved significantly the yield and time to fruiting. Daily variations in temperature did not markedly modify the yield. Significantly higher mushroom biomass was obtained with three wild European strains compared with three Brazilian cultivars. The very productive wild strain CA438-A gave mushrooms of size and dry matter content comparable to those of a cultivar. CONCLUSION: Commercial production of A. subrufescens can be developed in western countries on the wheat straw-based substrate commonly used for A. bisporus in these regions, by a simple modification of the casing mixture and maintaining the incubation temperature throughout the crop, which is expected to save energy during summer. Good yields were obtained cultivating European strains under optimised parameters.

Valorization of solid olive mill wastes by cultivation of a local strain of edible mushrooms.

Olive oil industry generates huge quantities of solid olive mill wastes (SOMW), causing environmental damage. Cultivation of edible mushrooms, such as Pleurotus ostreatus is a valuable approach for SOMW valorization. A local strain mycelium (Tizi-Ouzou, Algeria) of P. ostreatus (LPO) was isolated from castor oil plants. Oyster mushroom spawn, produced on barley grains, was used to inoculate wet SOMW, steamed in a traditional steamer during 45 min. The mycelium growth rate on SOMW was first estimated in Petri dish by measuring the surface colonized by the mycelium. The fruit body yields were estimated on culture bags containing 2 kg each of SOMW inoculated at 7% (w/w). The local strain potential was compared with that of a commercial one. Both strains produced high-quality mushrooms, but with low yields. The supplementation of the SOMW with wheat straw at the rate of 10% and 2% of CaCO3 had significantly enhanced the productivity of the two strains, multiplying it by 3.2 for LPO and by 2.6 for CPO.