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A number of experiments were done to further our understanding of the substrate utilization in button mushroom crops (Agaricus bisporus). An analysis of the degradation of dry matter of the substrate during a crop cycle revealed that for pin formation the upper layer is used, for the production of flush one all layers are involved and for flush two mainly the lower layer is used. A reduction in substrate depth leads to a decrease in yield/m2 but an apparent increase in yield per ton of substrate but with a lower mushroom quality. A short daily interruption of the connection between the casing soil with all the substrate results in a delay of the first flush. Repetitive interruption of the connection with all or only part of the substrate leads to a shift in yield from flush one to flush two but the total yield remains unchanged. The mycelial biomass in the substrate increases from filling up to pinning, a steeper increase during flush one, levelling off during flush two, indicating that in the period of venting and up to/including flush one, enzymes are secreted by growing hyphae generating nutrients to feed a fixed amount of mushroom biomass for two flushes. A sidewise extension of the substrate (without casing soil, thus not producing mushrooms) showed that the substrate at a distance more than 20-50 cm away from the casing soil does not contribute to feeding mushrooms in the first two flushes. The observations are discussed with respect to relevant previous research.ImportanceButton mushrooms (Agaricus bisporus) are cultivated on a substrate prepared of composted wheat straw, chicken and/or horst manure and gypsum. This manuscript describes a number of experiments indicating that in a harvesting strategy towards quality mushrooms there is a fixed amount of nutrients prepared/available to feed two flushes. The increase of mycelial biomass in the substrate after venting and especially during the development of flush one indicates that likely a branching of mycelium occurs during this period, leading to a substantial number of growing hyphal tips excreting lignocellulolytic enzymes that degrade substrate and make nutrients available during flush one which are utilised in flush one and two. The presently used substrate seems to have reached its limits and there are hardly opportunities for further improvement. Research on alternative, more energy dense, substrates are needed to improve the efficiency of the present production system.

The button mushroom Agaricus bisporus is represented mainly by two varieties, a secondarily homothallic variety with predominantly two heterokaryotic spores per basidia and a heterothallic variety with predominantly four homokaryotic spored basidium. Both varieties also differ in their recombination landscape with the former showing crossovers (CO) predominantly at chromosome ends whereas the latter has a more evenly distribution of CO over the chromosomes. The two varieties are compatible, and this has been used to study segregation of the basidial spore number (BSN) and the genomic positions of recombination, i.e., the CO landscape, in order to find the underlying genetic determinants. Knowledge on genes controlling CO positions might facilitate either the conservation of favorable allele combinations or the disruption of unwanted allele combinations to reduce linkage drag. For BSN, in total seven QTL were found with the major QTL on chromosome 1 explaining ca. 55% of the phenotypic variation. It appeared, however, difficult to map the recombination landscape. This phenotype can only be assessed in the meiotic offspring of an intervarietal hybrid which is a laborious and difficult task. Nevertheless, this was done, and we were able to map three QTLs for this trait, two on chromosome 1 and one on chromosome 2 not overlapping with the QTL for BSN. The hurdles encountered are discussed and a new strategy is proposed that can solves these. We propose to use two genetically unrelated mapping populations both offspring of a cross between a var. bisporus and a var. burnettii homokaryon and thus segregating both for CO and BSN. Homokaryotic offspring of both populations can be intercrossed without limitation of mating incompatibility and marker homozygosity and the hybrid mushrooms directly used to map BSN. Homokaryotic offspring of these hybrid mushrooms can be genotypes to assess CO positions using next generation sequencing technologies that will solve marker problems encountered, especially for genotyping chromosome ends. This new approach can be a useful strategy for a more efficient breeding strategy for mushrooms in general.

In heterothallic basidiomycete fungi, sexual compatibility is restricted by mating types, typically controlled by two loci: PR, encoding pheromone precursors and pheromone receptors, and HD, encoding two types of homeodomain transcription factors. We analysed the single mating-type locus of the commercial button mushroom variety, Agaricus bisporus var. bisporus, and of the related variety burnettii. We identified the location of the mating-type locus using genetic map and genome information, corresponding to the HD locus, the PR locus having lost its mating-type role. We found the mip1 and β-fg genes flanking the HD genes as in several Agaricomycetes, two copies of the β-fg gene, an additional HD2 copy in the reference genome of A. bisporus var. bisporus and an additional HD1 copy in the reference genome of A. bisporus var. burnettii. We detected a 140 kb-long inversion between mating types in an A. bisporus var. burnettii heterokaryon, trapping the HD genes, the mip1 gene and fragments of additional genes. The two varieties had islands of transposable elements at the mating-type locus, spanning 35 kb in the A. bisporus var. burnettii reference genome. Linkage analyses showed a region with low recombination in the mating-type locus region in the A. bisporus var. burnettii variety. We found high differentiation between β-fg alleles in both varieties, indicating an ancient event of recombination suppression, followed more recently by a suppression of recombination at the mip1 gene through the inversion in A. bisporus var. burnettii and a suppression of recombination across whole chromosomes in A. bisporus var. bisporus, constituting stepwise recombination suppression as in many other mating-type chromosomes and sex chromosomes.

BACKGROUND White rot fungi have been used to improve the nutritive value of lignocellulose for ruminants. In feed analysis, the Van Soest method is widely used to determine the cell wall contents. To assess the reliability of this method (Method A) for determination of cell wall contents in fungal‐treated wheat straw, we compared a combined monosaccharide analysis and pyrolysis coupled to gas chromatography with mass spectrometry (Py‐GC/MS) (Method B). Ruminal digestibility, measured as in vitro gas production (IVGP), was subsequently used to examine which method explains best the effect of fungal pretreatment on the digestibility of wheat straw. RESULTS Both methods differed considerably in the mass recoveries of the individual cell wall components, which changed on how we assess their degradation characteristics. For example, Method B gave a higher degradation of lignin (61.9%), as compared to Method A (33.2%). Method A, however, showed a better correlation of IVGP with the ratio of lignin to total structural carbohydrates, as compared to Method B (Pearson's r of −0.84 versus −0.69). Nevertheless, Method B provides a more accurate quantification of lignin, reflecting its actual modification and degradation. With the information on the lignin structural features, Method B presents a substantial advantage in understanding the underlying mechanisms of lignin breakdown. Both methods, however, could not accurately quantify the cellulose contents – among others, due to interference of fungal biomass. CONCLUSION Method A only accounts for the recalcitrant residue and therefore is more suitable for evaluating ruminal digestibility. Method B allows a more accurate quantification of cell wall, required to understand and better explains the actual modification of the cell wall. The suitability of both methods, therefore, depends on their intended purposes. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Oyster mushrooms are grown commercially worldwide, especially in many developing countries, for their easy cultivation and high biological efficiency. Pleurotus cornucopiae is one of the main oyster mushroom species because of its gastronomic value and nutraceutical properties. Cap color is an important trait, since consumers prefer dark mushrooms, which are now represented by only a small portion of the commercial varieties. Breeding efforts are required to improve quality-related traits to satisfy various demands of consumers. Here, we present a saturated genetic linkage map of P. cornucopiae constructed by using a segregating population of 122 monokaryons and 3,449 single nucleotide polymorphism (SNP) markers generated by the 2b-RAD approach. The map contains 11 linkage groups covering 961.6 centimorgans (cM), with an average marker spacing of 0.27 cM. The genome of P. cornucopiae was de novo sequenced, resulting in 425 scaffolds (>1,000 bp) with a total genome size of 35.1 Mb. The scaffolds were assembled to the pseudochromosome level with the assistance of the genetic linkage map. A total of 97% SNP markers (3,357) were physically localized on 140 scaffolds that were assigned to 11 pseudochromosomes, with a total of 32.5 Mb, representing 92.5% of the whole genome. Six quantitative trait loci (QTL) controlling cap color of P. cornucopiae were detected, accounting for a total phenotypic variation of 65.6%, with the highest value for the QTL on pseudochromosome 5 (18%). The results of our study provide a solid base for marker-assisted breeding for agronomic traits and especially for studies on biological mechanisms controlling cap color in oyster mushrooms. IMPORTANCE Oyster mushrooms are produced and consumed all over the world. Pleurotus cornucopiae is one of the main oyster mushroom species. Dark-cap oyster mushrooms are becoming more and more popular with consumers, but dark varieties are rare on the market. Prerequisites for efficient breeding programs are the availability of high-quality whole genomes and genetic linkage maps. Genetic studies to fulfill some of these prerequisites have hardly been done for P. cornucopiae. In this study, we de novo sequenced the genome and constructed a saturated genetic linkage map for P. cornucopiae. The genetic linkage map was effectively used to assist the genome assembly and identify QTL that genetically control the trait cap color. As well, the genome characteristics of P. cornucopiae were compared to the closely related species Pleurotus ostreatus. The results provided a basis for understanding the genetic background and marker-assisted breeding of this economically important mushroom species.

Degradation of lignocellulose by selective white-rot fungi can significantly improve the nutritional value of high lignocellulose containing biomass by affecting the access of rumen microbes to structural carbohydrates. To determine if such treated biomass is stable in time to allow it to be conserved for subsequent feeding to ruminant animals, wheat straw (WS) pre-treated for 7 weeks with either Ceriporiopsis subvermispora or Lentinula edodes was stored anaerobically up to 10 weeks at different temperatures (24.7–52.4 °C). Substrates were subsequently analysed for changes in pH, titratable acidity, fibre composition, in vitro gas production (IVGP) and colour, as well as polar metabolites by GC–MS and ceriporic acids by LC–MS. The increased titratable acidity of fungal treated WS during storage indicated acidification of the straw. A significant decrease in hemicellulose and an increase in acid detergent lignin content was observed at 52.4 °C. No negative effect of the storage condition on the degradability of both fungal treated WS in rumen fluid was observed. A darker colour was observed for substrates incubated at higher temperatures, coinciding with a strong accumulation of several organic acids and sugars. A decrease in ceriporic acid A, B, C and G produced by C. subvermispora was observed when stored at 52.4 °C from week 2–10. The results show that, although the chemical composition changes, anaerobic storage of fungal treated WS at different temperatures does not affect its fermentation potential for ruminants.

A variety of secondary metabolites are formed and compounds released during the bioconversion of lignocellulosic biomass by white-rot fungi that can affect the nutritional value and acceptance of the biomass by ruminants. Changes in pH, ergosterol content, fibre and metabolites composition of wheat straw (WS) incubated with either Ceriporiopsis subvermispora or Lentinula edodes for up to 8 weeks were investigated. With increases in mycelium content, significant decreases in absolute amount of hemicellulose, acid detergent lignin and, to a lesser extent, cellulose were observed in both fungal treatments. Acidification mainly occurred within the first four weeks of incubation, coinciding with the largest changes in metabolites profile. Diverse compounds, including organic acids and soluble sugars increased or decreased with C. subvermispora and L. edodes treatment. None of the thirty-four common mycotoxins analyzed were detected in WS after 8 weeks of fungal incubation. These results provide important information for application of fungal treated WS that might affect animal acceptance and performance.

Pleurotus ostreatus, one of the most widely cultivated edible mushrooms, produces high numbers of spores causing severe respiratory health problems for people, clogging of filters and spoilage of produce. A non-sporulating commercial variety (SPOPPO) has been successfully introduced into the market in 2006. This variety was generated by introgression breeding of a natural mutation into a commercial variety. Our cytological studies revealed that meiosis in the natural and derived sporeless strains was blocked in metaphase I, apparently resulting in a loss of spore formation. The gene(s) underlying this phenotype were mapped to an 80 kb region strongly linked to sporelessness and identified by transformation of wild type genes of this region into a sporeless strain. Sporulation was restored by re-introduction of the DNA sequence encoding the P. ostreatus meiotic recombination gene MSH4 homolog (poMSH4). Subsequent molecular analysis showed that poMSH4 in the sporeless P. ostreatus was interrupted by a DNA fragment containing a region encoding a CxC5/CxC6 cysteine cluster associated with Copia-type retrotransposons. The block of meiosis in metaphase I by a poMSH4 null mutant suggests that this protein plays an essential role in both Class I and II crossovers in mushrooms, similar to animals (mice), but unlike in plants. MSH4 was previously shown to be a target for breeding of sporeless varieties in P. pulmonarius, and the null mutant of the MSH4 homolog of S. commune (scMSH4) confers an extremely low level of spore formation. We propose that MSH4 homologs are likely to be a breeding target for sporeless strains both within Pleurotus sp. and in other Agaricales.

The successful application of fungal treated lignocellulosic biomass as an animal feed ingredient depends on its acceptance by animals. The objective of this study was to test the preference of fresh and stored fungal treated wheat straw (WS) by non-lactating Saanen goats. Three studies were conducted. Wheat straw was aerobically treated with Ceriporiopsis subvermispora (CS) and Lentinula edodes (LE) for 7 weeks before being stored at -20°C (fresh CS [CSf], fresh LE [LEf]) until use. The CSf and LEf were also anaerobically stored at 54°C (stored CS [CSs], stored LE [LEs]) for 7 weeks before being frozen at -20°C until use. In study 1, preference for individual feedstuffs was tested and included grass silage (GS), maize silage (MS), CSf, LEf and WS. Goats showed a strong preference for GS and were more reluctant to consume MS. Minor amounts of CSf, LEf and WS were consumed even when the preference time was extended to 2 h. Study 2 compared the preference of CSf, LEf and WS when included at 50% (as is basis) in a GS/MS based feed over 6 days. Goats had a higher (P < 0.05) intake rate of the CSf and LEf than the WS containing feed, with no significant difference observed between CSf and LEf. In study 3, the preference of goats between diets containing (50% as is basis) fresh and stored fungal treated WS was investigated. In an identical study design to study 2, the goats showed a higher intake rate for the CSs and LEs containing feeds. Wheat straw treated with C. subvermispora and L. edodes were significantly less preferred to GS and MS. This feeding study showed that fungal treated WS can be used as a major part in a diet for goat and the storage increases its palatability.

BACKGROUND: This study investigated the ruminal degradability of various wheat straw types by the white-rot fungi Ceriporiopsis subvermispora (CS) and Lentinula edodes (LE). Different cultivars (CV) of wheat straw at different maturity stages (MS) were treated with the fungi for 7 weeks and assessed for chemical composition and in vitro gas production (IVGP). RESULTS: Both fungi showed a more pronounced degradation of lignin on a more mature straw (MS3; 89.0%) in comparison with the straw harvested at an earlier stage (MS1; 70.7%). Quantitative pyrolysis coupled to gas chromatography and mass spectrometry, using 13C lignin as an internal standard 13C-IS Py-GC/MS revealed that lignin in more mature straw was degraded and modified to a greater extent. In contrast, cellulose was less degraded in MS3, as compared to MS1 (8.3% versus 14.6%). There was no effect of different MS on the IVGP of the fungus-treated straws. Among the different straw cultivars, the extent of lignin degradation varied greatly (47% to 93.5%). This may explain the significant (P

One of the main problems in the button mushroom industry is the rapid deterioration of fruit bodies after harvest. Today, nanotechnology has become a more reliable technique to improve the quality of products in food packaging. In the present study, the effectiveness of chitosan nanoparticles containing Citrus aurantium essential oil on postharvest quality of white button mushroom was examined and compared to essential oil fumigation and control treatments.; Results: Based on high-resolution transmission electron microscopy and dynamic light scattering, nanoparticles exhibited a spherical shape of 20-60 nm diameter. The results revealed that the application of chitosan nanoparticles loaded with C. aurantium oil significantly decelerated the rate of color change, weight loss and firmness compared to fumigation with essential oil and control treatments. Treatment of fruit bodies with chitosan nanoparticles loaded with C. aurantium oil promoted the accumulation of phenolic compounds and ascorbic acid, and resulted in increases in catalase and superoxide dismutase and a decrease in polyphenol oxidase activities, as the highest antioxidant capacity was observed after 15 days of cold storage.; Conclusion: This present research demonstrates that gradual release of C. aurantium essential oil from chitosan nanoparticles could be an effective and practical method for extending the shelf life of white button mushroom up to 15 days without significant decrease in antioxidant capacity. © 2018 Society of Chemical Industry.; © 2018 Society of Chemical Industry.

Background: No attention has been paid so far to the preservation of fungal-treated lignocellulose for longer periods. In the present study, we treated wheat straw (WS) with the white-rot fungi Ceriporiopsis subvermispora and Lentinula edodes for 8 weeks and assessed changes in pH, chemical composition and in vitro gas production (IVGP) weekly. Fungal-treated WS was also stored for 64 days 'as is', with the addition of lactic acid bacteria (LAB) or with a combination of LAB and molasses in airtight glass jars mimicking ensiling conditions.; Results: Both fungi significantly reduced the lignin and hemicellulose content of WS, and increased the cellulose content. The IVGP increased with increasing time of incubation, indicating the increase in digestibility. Both fungi lowered the pH of WS under 4.3, which guarantees an initial and stable low pH during anaerobic storage. Minor changes in fibre composition and IVGP were observed for stored L. edodes treated WS, whereas no change occurred for C. subvermispora.; Conclusion: It is possible to conserve C. subvermispora and L. edodes treated straw under anaerobic condition without additives up to 64 days. This finding is important for practical application to supply fungi-treated feed to ruminant animals for a prolonged period. © 2017 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.; © 2017 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Aim The present study evaluated differences between two strains of Ceriporiopsis subvermispora on improving the nutritive value and in vitro degradability of wheat straw. Methods and Results Wheat straw was treated with the fungi for 7 weeks. Weekly samples were analyzed for ergosterol content, in vitro gas production (IVGP), chemical composition and lignin degrading enzyme activity. Ergosterol data showed CS1 to have a faster initial growth than CS2 and reaching a stationary phase after 3 weeks. The IVGP of CS1-treated wheat straw exceeded the control earlier than CS2 (4 vs 5 weeks). CS1 showed a significantly higher (P < 0.001) selectivity in lignin degradation compared to CS2. Both strains showed peak activity of laccase and manganese peroxidase (MnP) at week 1. CS1 showed a significantly higher (P < 0.001) laccase activity, but lower (P = 0.008) MnP activity compared to CS2. Conclusion Both CS strains improved the nutritive value of wheat straw. Variation between strains was clearly demonstrated by their growth pattern and enzyme activities. Significance and Impact of Study The differences among the two strains provide an opportunity for future selection and breeding programs in improving the extent and selectivity of lignin degradation in agricultural biomass. This article is protected by copyright. All rights reserved.

The effect of ensiling and fungal treatment on the chemical composition and on in vitro gas production, an indication of rumen degradability, of maize stems was studied. Stems from two maize cultivars (LG30211 and MZP8057) were separated into three parts: upper (above internode 12 up to and including internode 15), middle (above internode 8 up to and including internode 11), and lower stem (above internode 5 up to and including internode 7). The three parts of the stems were ensiled for 1, 2, 4, and 8 weeks and the lower stem was treated with the white-rot fungi Lentinula edodes and Pleurotus eryngii for 3, 6, and 9 weeks. The dry matter content decreased and the acid detergent fibre (ADF) and acid detergent lignin (ADL) content increased from the top to the bottom in the stem. Upon ensiling, the neutral detergent fibre, ADF and ADL content increased. The gas production between 3 and 20 h, a measure of cell wall degradability in rumen fluid, did not change after ensiling, but gas production within 3 h and within 72 h decreased after ensiling. The acetic acid content and the proportion of ammonia-N in total N increased, and pH decreased, after 1 week of ensiling and did not show a clear trend from 1 to 8 weeks. The ADL and cellulose content of the lower part of the stem of both maize cultivars increased after fungal treatment and the hemicellulose content decreased except for the lower stem of MZP8057 treated with P. eryngii. The ratio of syringyl to guaiacyl compounds increased after fungal treatment. The gas production between 3 and 20 h and within 72 h decreased after fungal treatment. Both ensiling and fungal treatment cannot improve the degradability of the investigated maize stems.

The button mushroom Agaricus bisporus is an economically important crop worldwide. Many aspects of its cultivation are well known, except for the precise biological triggers for its fructification. By and large, for most basidiomycete species, nutrient availability, light and a drop in temperature are critical factors for fructification. A. bisporus deviates from this pattern in the sense that it does not require light for fructification. Furthermore its fructification seems to be inhibited by a self-generated factor which needs to be removed by microorganisms in order to initiate fruiting. This review explores what is known about the morphogenesis of fruiting initiation in A. bisporus, the microflora, the self-inhibitors for fruiting initiation and transcription factors involved. This information is subsequently contrasted with an overall model of the regulatory system involved in the initiation of the formation of primordia in basidiomycetes. The comparison reveals a number of the blank spots in our understanding of the fruiting process in A. bisporus.

A rapid and complete colonization of lignocellulosic biomass by white-rot fungi is a prerequisite for an efficient and contamination free delignification. To improve the colonization, mycelium growth and delignification by Ceriporiopsis subvermispora and Lentinula edodes of wheat straw, with addition of 2, 4, 6 and 10% of wheat bran for 0, 2, 4, 8 and 12 weeks, was evaluated. The ergosterol content, chemical composition and in vitro gas production (IVGP) by ruminal microorganisms were determined. The fungal biomass and starch amount was significantly (P < 0.001) affected by the inclusion level of wheat bran and incubation time. An increase in added wheat bran increased the fungal biomass with different effects observed for the two fungi. The added starch from the wheat bran was degraded by C. subvermispora, mainly during the first two weeks (up to 44%), whereas, L. edodes degraded starch during 12 weeks up to 83%. The supplementation of wheat bran, however, did not affect (P > 0.05) the amount of hemicellulose and lignin at the end of the incubation. There was no significant effect of wheat bran addition on the IVGP for the two fungal treatments. Supplementation of wheat straw with wheat bran resulted in a quicker fungal colonization, which is useful for C. subvermispora, a fungus that forms only thin mycelium and is more prone to contamination than L. edodes.

Background Pleurotus tuoliensis (Bailinggu) is a commercially cultivated mushroom species with an increasing popularity in China and other Asian countries. Commercial profits are now low, mainly due to a low yield, long cultivation period and sensitivity to diseases. Breeding efforts are thus required to improve agronomical important traits. Developing saturated genetic linkage and physical maps is a start for applying genetic and molecular approaches to accelerate the precise breeding programs. Results Here we present a genetic linkage map for P. tuoliensis constructed by using 115 haploid monokaryons derived from a hybrid strain H6. One thousand one hundred and eighty-two SNP markers developed by 2b–RAD (type IIB restriction-site associated DNA) approach were mapped to 12 linkage groups. The map covers 1073 cM with an average marker spacing of 1.0 cM. The genome of P. tuoliensis was de novo sequenced as 40.8 Mb and consisted of 500 scaffolds (>500 bp), which showed a high level of colinearity to the genome of P. eryngii var. eryngii. A total of 97.4% SNP markers (1151) were physically localized on 78 scaffolds, and the physical length of these anchored scaffolds were 33.9 Mb representing 83.1% of the whole genome. Mating type loci A and B were mapped on separate linkage groups and identified physically on the assembled genomes. Five putative pheromone receptors and two putative pheromone precursors were identified for the mating type B locus. Conclusions This study reported a first genetic linkage map integrated with physical mapping of the de novo sequenced genome and the mating type loci of an important cultivated mushroom in China, P. tuoliensis. The de novo sequenced and annotated genome, assembled using a 2b–RAD generated linkage map, provides a basis for marker-assisted breeding of this economic important mushroom species. Electronic supplementary material The online version of this article (10.1186/s12864-017-4421-z) contains supplementary material, which is available to authorized users.

Aim: In this study, the biological variation for improvement of the nutritive value of wheat straw by 12 Ceriporiopsis subvermispora, 10 Pleurotus eryngii and 10 Lentinula edodes strains was assessed. Screening of the best performing strains within each species was made based on the in vitro degradability of fungal-treated wheat straw. Methods and Results: Wheat straw was inoculated with each strain for 7 weeks of solid state fermentation. Weekly samples were evaluated for in vitro gas production (IVGP) in buffered rumen fluid for 72 h. Out of the 32 fungal strains studied, 17 strains showed a significantly higher (P < 0·05) IVGP compared to the control after 7 weeks (227·7 ml g−1 OM). The three best Ceriporiopsis subvermispora strains showed a mean IVGP of 297·0 ml g−1 OM, while the three best P. eryngii and L. edodes strains showed a mean IVGP of 257·8 and 291·5 ml g−1 OM, respectively. Conclusion: Ceriporiopsis subvermispora strains show an overall high potential to improve the ruminal degradability of wheat straw, followed by L. edodes and P. eryngii strains. Significance and Impact of the Study: Large variation exists within and among different fungal species in the valorization of wheat straw, which offers opportunities to improve the fungal genotype by breeding.

Besides their unique ability to depolymerize cell wall components, white-rot fungi are known to assimilate nitrogenous compounds from substrates. This modification may change protein solubility and fermentation in the rumen. To investigate this, the crude protein (CP) in fungal treated wheat straw (3 fungal species, 2 strains each) was fractioned according to the Cornell Net Carbohydrate and Protein System (CNCPS) and assessed for in vitro protein fermentation using a modified gas production technique (IVGPN). Results showed that fungi increased fraction A (instantaneously soluble CP; ∼2.6 times) and B1 (rapidly degradable; ∼1.2 times); and decreased the slowly degradable fraction B3 (∼41.6%) and unavailable fraction C (∼48.3%). The IVGPN of straw treated with Ceriporiopsis subvermispora strains were not different to the control, but increased by 30.2 to 47.1% in Pleurotus eryngii and Lentinula edodes strains. The IVGPN was significantly (P < 0.01) correlated to all fractions of CP, except fraction B1 and B2 (intermediately degradable). All fungi also increased the arginine (∼56%) and lysine (∼15%) contents. This study shows the importance of assessing the protein solubilization by different fungal strains, which can uncover unique mechanisms in the cell wall depolymerization.

Background The white-rot fungi Ceriporiopsis subvermispora (Cs), Pleurotus eryngii (Pe), and Lentinula edodes (Le) have been shown to be high-potential species for selective delignification of plant biomass. This delignification improves polysaccharide degradability, which currently limits the efficient lignocellulose conversion into biochemicals, biofuels, and animal feed. Since selectivity and time efficiency of fungal delignification still need optimization, detailed understanding of the underlying mechanisms at molecular level is required. The recently developed methodologies for lignin quantification and characterization now allow for the in-depth mapping of fungal modification and degradation of lignin and, thereby, enable resolving underlying mechanisms. Results Wheat straw treated by two strains of Cs (Cs1 and Cs12), Pe (Pe3 and Pe6) and Le (Le8 and Le10) was characterized using semi-quantitative py-GC–MS during fungal growth (1, 3, and 7 weeks). The remaining lignin after 7 weeks was quantified and characterized using 13C lignin internal standard based py-GC–MS and whole cell wall HSQC NMR. Strains of the same species showed similar patterns of lignin removal and degradation. Cs and Le outperformed Pe in terms of extent and selectivity of delignification (Cs ≥ Le >> Pe). The highest lignin removal [66% (w/w); Cs1] was obtained after 7 weeks, without extensive carbohydrate degradation (factor 3 increased carbohydrate-to-lignin ratio). Furthermore, though after treatment with Cs and Le comparable amounts of lignin remained, the structure of the residual lignin vastly differed. For example, Cα-oxidized substructures accumulated in Cs treated lignin up to 24% of the total aromatic lignin, a factor two higher than in Le-treated lignin. Contrarily, ferulic acid substructures were preferentially targeted by Le (and Pe). Interestingly, Pe-spent lignin was specifically depleted of tricin (40% reduction). The overall subunit composition (H:G:S) was not affected by fungal treatment. Conclusions Cs and Le are both able to effectively and selectively delignify wheat straw, though the underlying mechanisms are fundamentally different. We are the first to identify that Cs degrades the major β-O-4 ether linkage in grass lignin mainly via Cβ–O–aryl cleavage, while Cα–Cβ cleavage of inter-unit linkages predominated for Le. Our research provides a new insight on how fungi degrade lignin, which contributes to further optimizing the biological upgrading of lignocellulose.

True breeding of button mushrooms has hardly been done in the last decades, despite this species being one of the most cultivated mushrooms worldwide. Research done in the last 20 years has identified and characterised new germplasm and improved our understanding of the genetic base for some traits. A substantial collection of wild-collected strains is now available and partly characterised for a number of important traits such as disease resistance and yield. Most of the variations found in a number of important agronomic traits have a considerable heritability and are thus useful for breeding. Genetic marker technology has also developed considerably for this mushrooms in the last decade and used to identify quantitative trait loci (QTL) for important agronomic traits. This progress has, except for one example, not resulted so far into new commercially varieties. One of the reasons lies in the typical life cycle of the button mushroom Agaricus bisporus var. bisporus which hampers breeding. Joint investment is needed to solve technical problems in breeding. Special attention is needed for the protection of new varieties. Due to its typical life cycle, it is very easy to generate so called “look-a-likes” from protected cultivars by screening fertile single spore cultures. A consensus has been reached within the mushroom (breeding) industry to consider this method as the generation of essentially derived varieties as defined in plant breeding. Electronic supplementary material The online version of this article (doi:10.1007/s00253-017-8102-2) contains supplementary material, which is available to authorized users.

11 páginas.-- 4 figuras.-- 3 tablas.-- 32 referencias..-- Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biombioe.2017.07.003, Wheat straw and oak wood chips were incubated with Ceriporiopsis subvermispora and Lentinula edodes for 8 weeks. Samples from the fungal treated substrates were collected every week for chemical characterization. L. edodes continuously grew during the 8 weeks on both wheat straw and oak wood chips, as determined by the ergosterol mass fraction of the dry biomass. C. subvermispora colonized both substrates during the first week, stopped growing on oak wood chips, and resumed growth after 6 weeks on wheat straw. Detergent fiber analysis and pyrolysis coupled to gas chromatography/mass spectrometry showed a selective lignin degradation in wheat straw, although some carbohydrates were also degraded. L. edodes continuously degraded lignin and hemicelluloses in wheat straw while C. subvermispora degraded lignin and hemicelluloses only during the first 5 weeks of treatment after which cellulose degradation started. Both fungi selectively degraded lignin in wood chips. After 4 weeks of treatment, no significant changes in chemical composition were detected. In contrast to L. edodes, C. subvermispora produced alkylitaconic acids during fungal treatment, which paralleled the degradation and modification of lignin indicating the importance of these compounds in delignification. Light microscopy visualized a dense structure of wood chips which was difficult to penetrate by the fungi, explaining the relative lower lignin degradation compared to wheat straw measured by chemical analysis. All these changes resulted in an increased in in vitro rumen degradability of wheat straw and oak wood chips. In addition, more glucose and xylose were released after enzymatic saccharification of fungal treated wheat straw compared to untreated material., This research was part of a large program entitle: “Waste to Resource” and this was supported by the Dutch Technology Foundation (STW), which is part of the Netherlands Organization for Scientific Research (NWO), which is partly funded by the Dutch Ministry of Economic Affairs. This specific research reported here was part of the project entitled: “Increasing the utilization of organic waste and low value feeds with the help of lignin degrading fungi” (project number 111611). This particular research was co-sponsored by Agrifirm, Purac, DSM, Den Ouden, Hofmans, the Dutch commodity boards for dairy and horticulture, and Wageningen University. We would like to thank Agrifirm for analyzing part of the samples for dry matter, ash, crude protein, NDF and ADF. We would like to thank Dyadic Nederland BV for supplying the CMAX3 enzyme mixture. This study has also partially been funded by the Spanish projectsAGL2011-25379, AGL2014-53730-R and CTQ2014-60764-JIN (co-financed by FEDER funds), the CSIC project2014-40E-097 and the EU-project INDOX (KBBE-2013-7-613549).

BACKGROUND Food is a potential source of immunomodulating compounds that may be used to steer immune responses towards a desired status such as reducing inflammatory disorders. However, to identify and characterize such bioactive compounds, biologically relevant and standardized assays are required. Macrophages play an important role in immunomodulation and are suited for developing cell-based assays. An assay was developed based on macrophages, in a homogeneous differentiation state, using the human monocytic cell line THP-1 previously used to assess immunomodulatory properties of low-molecular-weight allergens, hormones, dietary supplements and therapeutic drugs. RESULTS Zymosan and mushroom polysaccharide extracts lead to a heterogeneous differentiation state of THP-1 monocytes, and these cells secrete low levels of cytokines upon stimulation. Differentiation into macrophages using a low concentration of phorbol 12-myristate 13-acetate improved responsiveness. Elevated levels of cytokines were secreted by cells in a homogenous differentiation state. In addition, it was determined that the assay performs best when using cells at a concentration of (2.5–5)?×?105 cells mL-1. CONCLUSION An assay was developed suitable to distinguish the immunomodulatory properties of food compounds in a reproducible manner. It was evaluated using eight mushroom species by measuring the secretion of relevant cytokines TNF-a, IL-1s, IL-6 and IL-10. © 2014 Society of Chemical Industry

The aim of this study was optimizing Ceriporiopsis subvermispora and Lentinula edodes pre-treatment of wheat straw and wood chips by adding urea, manganese and linoleic acid. Optimization was defined as more lignin degradation and an increase in in vitro gas production (IVGP), which is a model for rumen degradation, in comparison to fungal treatment without additives. First urea, manganese and linoleic acid were added separately to C. subvermispora or L. edodes treatment of wheat straw and wood chips. Only manganese and linoleic acid addition improved lignin degradation and IVGP compared to the same treatment without additives. Mn (150 μg/g wheat straw) influenced C . subvermispora treatment most. A combination of manganese and linoleic acid was also applied since both act on manganese peroxidase. This combination did indeed increase lignin degradation in wheat straw by C . subvermispora , but IVGP was not changed. None of the additions had a significant effect on the other fungus–substrate combinations tested here.

The demand for button mushrooms of high quality is increasing. Superior button mushroom varieties require the combination of multiple traits to maximize productivity and quality. Very often these traits are correlated and should, therefore, be evaluated together rather than as single traits. In order to unravel the genetic architecture of multiple traits of Agaricus bisporus and the genetic correlations among traits, we have investigated a total of six agronomic and quality traits through multi-trait QTL analyses in a mixed-model. Traits were evaluated in three heterokaryon sets. Significant phenotypic correlations were observed among traits. For instance, earliness (ER) correlated to firmness (FM), cap color, and compost colonization, and FM correlated to scales (SC). QTLs of different traits located on the same chromosomes genetically explains the phenotypic correlations. QTL detected on chromosome 10 mainly affects three traits, i.e., ER, FM and SC. It explained 31.4 % phenotypic variation of SC on mushroom cap (heterokaryon Set 1), 14.9 % that of the FM (heterokaryon Set 3), and 14.2 % that of ER (heterokaryon Set 3). High value alleles from the wild parental line showed beneficial effects for several traits, suggesting that the wild germplasm is a valuable donor in terms of those traits. Due to the limitations of recombination pattern, we only made a start at understanding the genetic base for several agronomic and quality traits in button mushrooms. Electronic supplementary material The online version of this article (doi:10.1186/s13568-016-0239-3) contains supplementary material, which is available to authorized users.

International audience; The button mushroom (Agaricus bisporus) is one of the world's most cultivated mushroom species, but in spite of its economic importance generation of new cultivars by outbreeding is exceptional. Previous genetic analyses of the white bisporus variety, including all cultivars and most wild isolates revealed that crossing over frequencies are low, which might explain the lack of introducing novel traits into existing cultivars. By generating two high quality whole genome sequence assemblies (one de novo and the other by improving the existing reference genome) of the first commercial white hybrid Horst U1, a detailed study of the crossover (CO) landscape was initiated. Using a set of 626 SNPs in a haploid offspring of 139 single spore isolates and whole genome sequencing on a limited number of homo- and heterokaryotic single spore isolates, we precisely mapped all COs showing that they are almost exclusively restricted to regions of about 100kb at the chromosome ends. Most basidia of A. bisporus var. bisporus produce two spores and pair preferentially via non-sister nuclei. Combined with the COs restricted to the chromosome ends, these spores retain most of the heterozygosity of the parent thus explaining how present-day white cultivars are genetically so close to the first hybrid marketed in 1980. To our knowledge this is the first example of an organism which displays such specific CO landscape.

Mushrooms are well known for their immunomodulating capacities. However, little is known about how mushroom-stimulated dendritic cells (DCs) affect T cells. Therefore, we investigated the effect of mushroom compounds derived from seven edible mushroom species on DCs, their fate in DCs, and the effect of the mushroom-stimulated DCs on T cells. Each mushroom species stimulated DCs in a different manner as was revealed from the DC’s cytokine response. Assessing DC maturation revealed that only one mushroom species, Agaricus subrufescens, induced complete DC maturation. The other six mushroom species upregulated MHC-II and CD86 expression, but did not significantly affect the expression of CD40 and CD11c. Nevertheless, mushroom compounds of all investigated mushroom species are endocytosed by DCs. Endocytosis is most likely mediated by C-type lectin receptors (CLRs) because CLR binding is Ca2+ dependent, and EGTA reduces TNF-α secretion with more than 90%. Laminarin partly inhibited TNF-α secretion indicating that the CLR dectin-1, among other CLRs, is involved in binding mushroom compounds. Stimulated DCs were shown to stimulate T cells; however, physical contact of DCs and T cells is not required. Because CLRs seem to play a prominent role in DC stimulation, mushrooms may function as a carbohydrate containing adjuvant to be used in conjunction with anti-fungal vaccines.

The collection of Plant Breeding contains a large number of strains of Pleurotus eryngii and Lentinula edodes, two fungi producing edible mushrooms and also known by their selective degradation of lignin in lignocellulosic materials.

Mushrooms are prone to develop brown discolouration due to bruising caused by mechanical damage during harvest, which leads to reduced quality of the mushrooms. In order to study the mechanism behind discolouration and to breed for bruise-related browning resistant strains, a high throughput bruise application method and reliable quantification of browning sensitivity is essential. In this study a new bruising device was developed to bruise mushrooms in a fast and reproducible way. The bruising device can apply damage to the cap tissue of button mushrooms by a slip-shear sliding process. The severity of the bruise was quantified with a computer imaging system combined with a newly developed software programme. A protocol was developed to obtain the most reliable and reproducible method to compare browning sensitivity of different Agaricus bisporus strains. For this purpose, the extent of damage applied, time between harvest and bruising, developmental stage of the mushrooms and flushes were taken into account. The new method of bruising and image based quantification was subsequently applied to a collection of wild, commercial and hybrid A. bisporus strains and a distinction could be made between bruise-related browning sensitive mushrooms and tolerant mushrooms

Using degenerated primers for conserved regions of Rec A homologs we have isolated a gene from Pleurotus ostreatus that shows characteristic features of RAD51 homologs. The encoded amino acid sequence of P. ostreatus RAD51 ( PoRAD51 ) shows greatest sequence similarities with RAD51 from Coprinus cinereus (89% identity). Furthermore the genomic organisation of PoRAD51 is almost identical to that of RAD51 from C. cinereus. Northern analysis shows that the expression of PoRAD51 is found in vegetative mycelium, and fruit body tissue, and that it is expressed at elevated levels in lamellae/basidia and following DNA damage. A sporulation deficient mutant strain of P. ostreatus (ATTC 58937) showed expression patterns of the RAD51 gene that are similar those of the normal sporulating strain.

In ruminant nutrition, there is an increasing interest for ingredients that do not compete with human nutrition. Ruminants are specialists in digesting carbohydrates in plant cell walls; therefore lignocellulosic biomass has potential in ruminant nutrition. The presence of lignin in biomass, however, limits the effective utilization of cellulose and hemicellulose. Currently, most often chemical and/or physical treatments are used to degrade lignin. White rot fungi are selective lignin degraders and can be a potential alternative to current methods which involve potentially toxic chemicals and expensive equipment. This review provides an overview of research conducted to date on fungal pretreatment of lignocellulosic biomass for ruminant feeds. White rot fungi colonize lignocellulosic biomass, and during colonization produce enzymes, radicals and other small compounds to breakdown lignin. The mechanisms on how these fungi degrade lignin are not fully understood, but fungal strain, the origin of lignocellulose and culture conditions have a major effect on the process. Ceriporiopsis subvermispora and Pleurotus eryngii are the most effective fungi to improve the nutritional value of biomass for ruminant nutrition. However, conclusions on the effectiveness of fungal delignification are difficult to draw due to a lack of standardized culture conditions and information on fungal strains used. Methods of analysis between studies are not uniform for both chemical analysis and in vitro degradation measurements. In vivo studies are limited in number and mostly describing digestibility after mushroom production, when the fungus has degraded cellulose to derive energy for fruit body development. Optimization of fungal pretreatment is required to shorten the process of delignification and make it more selective for lignin. In this respect, future research should focus on optimization of culture conditions and gene expression to obtain a better understanding of the mechanisms involved and allow the development of superior fungal strains to degrade lignin in biomass.

A 300-bp repetitive element was found in the genome of the white button mushroom, Agaricus bisporus , and designated Abr1 . It is present in ∼15 copies per haploid genome in the commercial strain Horst U1. Analysis of seven copies showed 89 to 97% sequence identity. The repeat has features typical of class II transposons (i.e., terminal inverted repeats, subterminal repeats, and a target site duplication of 7 bp). The latter shows a consensus sequence. When used as probe on Southern blots, Abr1 identifies relatively little variation within traditional and present-day commercial strains, indicating that most strains are identical or have a common origin. In contrast to these cultivars, high variation is found among field-collected strains. Furthermore, a remarkable difference in copy numbers of Abr1 was found between A. bisporus isolates with a secondarily homothallic life cycle and those with a heterothallic life cycle. Abr1 is a type II transposon not previously reported in basidiomycetes and appears to be useful for the identification of strains within the species A. bisporus .

We investigated the patterns of mitochondrial DNA variation in the global population ofthe commercial mushroom Agaricus bisporus . Through the analysis of RFLP’s among 441isolates from nine countries in North America and Eurasia, we found a total of 140mtDNA haplotypes. Based on population genetic analysis, there are four genetically dis-tinct natural populations in this species, found in coastal California, desert California,France and Alberta (Canada). While 134 of the 140 mtDNA haplotypes were unique to sin-gle geographical regions, two mtDNAhaplotypes, mt001 and mt002, were found in almostevery population surveyed. These two mtDNA haplotypes also predominate among culti-vars used throughout the world for at least the last two decades. These two mtDNA haplo-types are more similar to the cosmopolitan groups of mtDNA haplotypes than to theindigeneous clusters of mtDNA haplotypes from the two Californian regions. Keywords : artificial dispersal, genetic polymorphism, germ plasm, population structure

We studied the appearance of fruit body primordia, the growth of individual fruit bodies and the development of the consecutive flushes of the crop. Relative growth, measured as cap expansion, was not constant. It started extremely rapidly, and slowed down to an exponential rate with diameter doubling of 1.7 d until fruit bodies showed maturation by veil breaking. Initially many outgrowing primordia were arrested, indicating nutritional competition. After reaching 10 mm diameter, no growth arrest occurred; all growing individuals, whether relatively large or small, showed an exponential increase of both cap diameter and biomass, until veil breaking. Biomass doubled in 0.8 d. Exponential growth indicates the absence of competition. Apparently there exist differential nutritional requirements for early growth and for later, continuing growth. Flushing was studied applying different picking sizes. An ordinary flushing pattern occurred at an immature picking size of 8 mm diameter (picking mushrooms once a day with a diameter above 8 mm). The smallest picking size yielded the highest number of mushrooms picked, confirming the competition and arrested growth of outgrowing primordia: competition seems less if outgrowing primordia are removed early. The flush duration (i.e. between the first and last picking moments) was not affected by picking size. At small picking size, the subsequent flushes were not fully separated in time but overlapped. Within 2 d after picking the first individuals of the first flush, primordia for the second flush started outgrowth. Our work supports the view that the acquisition of nutrients by the mycelium is demand rather than supply driven. For formation and early outgrowth of primordia, indications were found for an alternation of local and global control, at least in the casing layer. All these data combined, we postulate that flushing is the consequence of the depletion of some unknown specific nutrition required by outgrowing primordia.

Repetitive DNA elements are ubiquitous constituents of eukaryotic genomes. The biological roles of these repetitive elements, supposed to impact genome organization and evolution, are not completely elucidated yet. The availability of whole genome sequence offers the opportunity to draw a picture of the genome-wide distribution of these elements and provide insights into potential mechanisms of genome plasticity. The present study uses in silico approaches to describe tandem repeats and transposable elements distribution in the genome of the button mushroom, Agaricus bisporus . Transposable elements comprised 12.43% of the assembled genome, and 66% of them were found clustered in the centromeric or telomeric regions. Methylation of retrotransposon has been demonstrated. A total of 1996 mini-, 4062 micro-, and 37 satellites motifs were identified. The microsatellites appeared widely and evenly spread over the whole genome sequence, whereas the minisatellites were not randomly distributed. Indeed, minisatellites were found to be associated with transposable elements clusters. Telomeres exhibited a specific sequence with a T n AG n signature. A comparison between the two available genome sequences of A. bisporus was also performed and sheds light on the genetic divergence between the two varieties. Beyond their role in genome structure, repeats provide a virtually endless source of molecular markers useful for genetic studies in this cultivated species.

The genome of the cultivated basidiomycete Agaricus bisporus Horst U1 and of its homokaryotic parents has been characterized by using an optimized method of pulsed-field gel electrophoresis. Expressed sequence tags obtained as expressed cDNAs from a primordial tissue-derived cDNA library and a number of previously isolated genes were used to identify the individual chromosomes of the parental lines of Horst U1. The genome consists of 13 chromosomes, and its total size is 31 Mb. For those chromosomes that could not be resolved by contour-clamped homogeneous electric field electrophoresis, the segregation of marker genes was studied in a set of 86 homokaryotic offspring of Horst U1. At least two markers were assigned to each individual chromosome. In this way all individual chromosomes were unequivocally identified. The large size difference observed between the homologous chromosomes IX, harboring the rDNA repeat, was shown to be largely due to a higher copy number of rDNA in parental strain H97 than in parental strain H39.

Horgen, P. A, Carvalho, D., Sonnenberg, A., Li, A., and Van Griensven, L. J. L. D. 1996. Chromosomal abnormalities associated with strain degeneration in the cultivated mushroom,Agaricus bisporus. Fungal Genetics and Biology20,299–241. Commercially grown strains of the button mushroom,Agaricus bisporus,on occasion produce sectors that display undesirable phenotypes. Such sectors show altered compost colonization, have reduced yield, and produce inferior quality mushrooms. The current study compared isolates of eight irreversible sectors to the original cultivator (U1) from which they arose. Gene-specific and anonymous DNA probes were used to identify RFLPs and chromosomal differences (CHEF analyses) between the U1 sectors and the normal U1 cultivar. A number of differences were noted including loss of heterozygosity at specific loci, deheterokaryotization, somatic recombination, chromosomal loss, chromosomal length polymorphisms, possible chromosomal translocations, and changes in copy number of the ribosomal DNA repeat.

The gene encoding NADP+-dependent glutamate dehydrogenase (gdhA) was isolated from an Agaricus bisporus recombinant phage lambda library. The deduced amino acid sequence would specify a 457-amino acid protein that is highly homologous in sequence to those derived from previously isolated and characterized genes coding for microbial NADP+-GDH. The open reading frame is interrupted by six introns. None of the introns is located at either one of the positions of the two introns conserved in the corresponding open reading frames of the ascomycete fungi Aspergillus nidulans and Neurospora crassa. Northern analysis suggests that the A. bisporus gdhA gene is transcriptionally regulated and that, unlike the case in ascomycetes, transcription of this gene is repressed upon the addition of ammonium to the culture.

Differential screening of a cDNA library was used to clone genes that are specifically expressed during mushroom development in the basidiomycete Agaricus bisporus. One of the isolated genes encodes a polypeptide of 112 amino acid residues and belongs to the fungal gene family encoding hydrophobins. This gene, hypA, has the characteristic pattern of eight cysteine residues at conserved positions and a hydrophobicity pattern that is very similar to class I hydrophobins. Elucidation of the genomic structure of hypA led to the identification of a second copy, hypC, located downstream of hypA. Although at a much lower level, hypC is like hypA specifically expressed on fruit bodies. The hypA mRNA level is transiently increased ten days after fruit body induction and expression appears to be associated with rapid expansion of the mushroom caps. In mushroom caps, very high concentrations of hypA messengers were found in the (outer) peel tissue, where they accumulate to more than 60% of the total mRNA mass. The corresponding protein with a molecular mass of 8 to 9 kDa was purified from this peel tissue and was identified by N-terminal sequencing. Our results suggest that HYPA forms a protective hydrophobic layer instrumental in cap formation.

Maize stover, rice straw, oil palm fronds and sugarcane bagasse were treated with the white-rot fungi Ceriporiopsis subvermispora, Lentinula edodes, Pleurotus eryngii, or Pleurotus ostreatus at 24 °C for 0–6 weeks. The fungi increased total gas production from oil palm fronds by 68–132%, but none of the fungi improved the in vitro rumen fermentability of maize stover. C. subvermispora and L. edodes increased total gas production of sugarcane bagasse by 65–71%, but P. eryngii and P. ostreatus decreased it by 22–50%. There was a linear relationship (P

Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the “button mushroom” forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost and during mushroom formation. The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation are more highly expressed in compost. The striking expansion of heme-thiolate peroxidases and β-etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor . A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics.

Mushrooms are known for their immune-modulating and anti-tumour properties. The polysaccharide fraction, mainly beta-glucans, is responsible for the immune-modulating effects. Fungal beta-glucans have been shown to activate leukocytes, which depend on structural characteristics of beta-glucans. As edible mushrooms come in contact with the intestinal immune system, effects on enterocytes are also interesting. Our aim was to evaluate the effect of mushroom polysaccharide extracts varying in beta-glucan structure on nitric oxide production by bone marrow-derived macrophages (BMMs) from mice and on nuclear factor-kappaB transactivation in human intestinal Caco-2 cells. We demonstrated that extracts from Agaricus bisporus stimulated nitric oxide production by BMM, whereas extracts from Coprinus comatus and spores of Ganoderma lucidum had only minor effects. Furthermore, extracts of A. blazei Murill and Phellinus linteus had no effect at all. Almost all mushroom extracts lowered nuclear factor-kappaB transactivation in Caco-2 cells. Structural analysis of A. bisporus compared with A. blazei Murill suggests that branching of the beta-glucan chain is essential for immune-stimulating activity. In conclusion, extracts from A. bisporus activate BMM, without activating enterocytes. These characteristics make A. bisporus an attractive candidate as a nutritional compound to stimulate the immune response in depressed states of immunity.

Fruiting initiation in mushrooms can be triggered by a variety of environmental and biochemical stimuli, including substances of natural or synthetic origin. In this work ostreolysin, a cytolytic protein specifically expressed during the formation of primordia and fruit bodies of Pleurotus ostreatus, was applied to nutrient media inoculated with mycelium of P. ostreatus, and its effects on mycelial growth and fructification of the mushroom studied. The addition of ostreolysin slightly inhibited the growth of mycelium, but strongly induced the formation of primordia, which appeared 10 d earlier than in control plates supplemented with bovine serum albumin or with the dissolving buffer alone. Moreover, ostreolysin stimulated the subsequent development of primordia into fruit bodies. However, direct involvement of this protein in the sporulation of the mushroom is unlikely, as it was also detected in large amounts in the non-sporulating strain of P. ostreatus.

Background - We investigate the scope for selection at the level of nuclei within fungal individuals (mycelia) of the mutualistic Termitomyces cultivated by fungus-growing termites. Whereas in most basidiomycete fungi the number and kind of nuclei is strictly regulated to be two per cell, in Termitomyces mycelia the number of nuclei per cell is highly variable. We hypothesised that natural selection on these fungi not only occurs between mycelia, but also at the level of nuclei within the mycelium. We test this hypothesis using in vitro tests with five nuclear haplotypes of a Termitomyces species. Results - First, we studied the transition from a mixture of five homokaryons (mycelia with identical nuclei) each with a different nuclear haplotype to heterokaryons (mycelia with genetically different nuclei). In vitro cultivation of this mixture for multiple asexual transfers led to the formation of multiple heterokaryotic mycelia, and a reduction of mycelial diversity over time. All heterokaryotic mycelia contained exactly two types of nucleus. The success of a heterokaryon during in vitro cultivation was mainly determined by spore production and to a lesser extent by mycelial growth rate. Second, heterokaryons invariably produced more spores than homokaryons implying that homokaryons will be outcompeted. Third, no homokaryotic ‘escapes’ from a heterokaryon via the formation of homokaryotic spores were found, despite extensive spore genotyping. Fourth, in contrast to most studied basidiomycete fungi, in Termitomyces sp. no nuclear migration occurs during mating, limiting the scope for nuclear competition within the mycelium. Conclusions - Our experiments demonstrate that in this species of Termitomyces the scope for selection at the level of the nucleus within an established mycelium is limited. Although ‘mate choice’ of a particular nuclear haplotype is possible during mating, we infer that selection primarily occurs between mycelia with two types of nucleus (heterokaryons).

Here we describe the isolation of a Pleurotus ostreatus gene PoDMC1. The predicted amino acid sequence of the oyster mushroom gene is 62% identical to the yeast DMC1 and 60% identical to human DMC1. The highest degree of amino acid identity (88%), however, was shown with Coprinus CoLIM15, a DMC1 homolog recently found in Coprinus cinereus. The exact matching of sizes and positions of most introns in both basidiomycete genes underlines the close relationship between these DMC1 orthologs. The RecA homolog DMC1 from yeast and its orthologs from other species have been reported to be meiosis specific and essential for sporulation. Here we show that PoDMC1 is exclusively expressed in the lamellae/basidiospore fraction of fruit bodies and not in somatic cells of fruiting bodies or in vegetative mycelium. Furthermore, the gene is not expressed in the lamellae/basidiospore fraction of a nonsporulating mutant of P. ostreatus. Since one of the major problems in cultivating the oyster mushroom is the abundant sporulation that causes allergic reactions in man, PoDMC1 could be an important target gene in constructing sporeless Pleurotus strains.

AB: A repeated sequence containing a microsatellite core sequence was found and characterized in the A. bisporus genome. A 16-mer oligonucleotide (TATG)4 primer based on this microsatellite core sequence led to polymerase chain reaction (PCR) products generated by directed amplification of microsatellite-region DNA (DAMD). The DAMD-PCR products allowed the study of the distribution of microsatellite in the genome of four distinct A. bisporus industrial strains (SC 940205, SC 960401, SC 930503, and SC970301), A. bisporus H97 line, and Pleurotus species (P. pulmonarius SC 920542, P. sajor-caju SC 971206 and P. florida SC 971203). The study demonstrated the reproducibility of the patterns obtained with the DAMD-PCR products by the lack of variation between the patterns obtained with 2 DNA extracts of each strain. Hence, the TATG4 16-mer primer is useful not only as a marker of the A. bisporus genome, allowing an estimation of interstrain variability, but also as a reliable and easy fingerprinting method for wild and industrial A. bisporus strains. This is thought to be the first microsatellite reported in cultivated edible mushrooms

Compost is the preferred substrate for growth of the edible fungus Agaricus bisporus .Utilization of compost requires the production of enzymes involved in degradation of lignocellulolytic components.For molecular characterization of these processes we are isolating the encoding genes.By applying heterologous screening techniques, we have cloned such a gene, which is specifically induced on compost encoding an endo-1,4-β-xylanase ( xlnA ) belonging to glycosyl hydrolase family 10.The gene encodes a pre-protein of 333 amino acid residues with a predicted molecular mass of 34,946 for the mature protein.The open reading frame is interrupted by ten introns of which introns 5 and 6 are separated by an exon of only two base-pairs. High expression of the xlnA gene was observed in vegetative mycelium grown on sterilized compost while xlnA messengers were not detected in fruit bodies. Addition of glucose or xylose to compost repressed xlnA expression. When glucose-grown colonies were transferred to a medium containing cellulose, xylan or xylose as sole carbon source, the organism responded by expressing xlnA at a high level for a short period. Transfer from glucose to compost yielded a much stronger and constant xlnA induction. A similar pattern of expression was found for the cel3 gene encoding a cellulase, suggesting that these genes are induced by compost-specific factors rather than by the substrates they act upon. Antiserum raised against XLNA protein, which was heterologously expressed in Escherichia coli , detected, when the fungus was grown on compost, an extracellular protein of 33 kDa with endo-xylanase activity.

We evaluated the influence of mitochondrial haplotype on growth of the common button mushroom Agaricus bisporus. Ten pairs of heterokaryon strains, each pair having the same nuclear genome but different mitochondrial genomes, were produced by controlled crosses among a group of homokaryons of both wild and commercial origins. Seven genetically distinct mitochondrial DNA (mtDNA) haplotypes were evaluated in different nuclear backgrounds. The growth of heterokaryon pairs differing only in their mtDNA haplotypes was compared by measuring mycelial radial growth rate on solid complete yeast medium (CYM) and compost extract medium and by measuring mycelial dry weight accumulation in liquid CYM. All A. bisporus strains were incubated at temperatures similar to those utilized in commercial production facilities (18, 22, and 26(deg)C). Statistically significant differences were detected in 8 of the 10 heterokaryon pairs evaluated for one or two of the three growth parameters measured. Some heterokaryon pairs showed differences in a single growth parameter at all three temperatures of incubation, suggesting a temperature-independent difference. Others showed differences at only a single temperature, suggesting a temperature-dependent difference. The influence of some mtDNA haplotypes on growth was dependent on the nuclear genetic background. Our results show that mtDNA haplotype can influence growth of A. bisporus heterokaryons in some nuclear backgrounds. These observations demonstrate the importance of including a number of mitochondrial genotypes and evaluating different nuclear-mitochondrial combinations of A. bisporus in strain improvement programs.