J.J.P. Baars

Fungal treated lignocellulosic biomass as ruminant feed ingredient: a review.

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.

Effects of mushroom‐derived β‐glucan‐rich polysaccharide extracts on nitric oxide production by bone marrow‐derived macrophages and nuclear factor‐κB transactivation in Caco‐2 reporter cells: Can effects be explained by structure?

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.

Nitrogen assimilating enzymes in the white button mushroom Agaricus bisporus

Agaricus bisporus has the enzymic potential to assimilate ammonia by the activities of glutamine synthetase (EC 6.3.1.2), NADp-dependent glutamate dehydrogenase (EC 1.4.1.2) and NADP-dependent glutamate dehydrogenase (EC 1.4.1.4). It also contains glutamate synthase (EC 1.4.7.1) and a number of transaminating activities like glutamate-oxaloacetate transaminase (EC 2.6.1.1), glutamate-pyruvate transaminase (EC 2.6.1.2) and alanine-glyoxylate transaminase (EC 2.6.1.44). A. bisporus showed good growth in a defined buffered medium on glucose as a carbon source and a number of organic nitrogen compounds or ammonia as a nitrogen source. No growth was observed using nitrate as a nitrogen source. A. bisporus was not able to use organic nitrogen containing substances as a sole nitrogen and carbon source. Specific activities of the ammonia assimilating enzymes showed some variation when mycelia were cultivated on different nitrogen sources. Highest specific activities for glutamine synthetase, NAD-dependent glutamate dehydrogenase and NADP-dependent glutamate dehydrogenase were found when mycelia were grown on glutamate as a nitrogen source. Lowest values were found when the mycelia were grown on ammonia or glutamine. The specific activities of the ammonia assimilating enzymes showed no variation during maturation of the sporophores.

Nucleotide sequence and expression of the gene encoding NADP(+) dependent glutamate dehydrogenase (gdhA) from Agaricus bisporus

The gene encoding NADP+-dependent glutamate dehydrogenase (gdhA) was isolated from anAgaricus bisporus recombinant phageλ 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 fungiAspergillus nidulans andNeurospora crassa. Northern analysis suggests that theA. 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.

Effect of fungal treatments of fibrous agricultural by-products on chemical composition and in vitro rumen fermentation and methane production

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<0.05) between the proportion of lignin in the original substrate and the increase in in vitro gas production observed for C. subvermispora and L. edodes treatments (R2=0.92 and 0.96, respectively). It is concluded that C. subvermispora and L. edodes have a particularly high potential to improve the nutritive value of highly lignified ruminant feeds.

Lecanicillium fungicola: causal agent of dry bubble disease in white-button mushroom.

UNLABELLED Lecanicillium fungicola causes dry bubble disease in commercially cultivated mushroom. This review summarizes current knowledge on the biology of the pathogen and the interaction between the pathogen and its most important host, the white-button mushroom, Agaricus bisporus. The ecology of the pathogen is discussed with emphasis on host range, dispersal and primary source of infection. In addition, current knowledge on mushroom defence mechanisms is reviewed. TAXONOMY Lecanicillium fungicola (Preuss) Zare and Gams: Kingdom Fungi; Phylum Ascomycota; Subphylum Pezizomycotina; Class Sordariomycetes; Subclass Hypocreales; Order Hypocreomycetidae; Family Cordycipitaceae; genus Lecanicillium. HOST RANGE Agaricus bisporus, Agaricus bitorquis and Pleurotus ostreatus. Although its pathogenicity for other species has not been established, it has been isolated from numerous other basidiomycetes. DISEASE SYMPTOMS Disease symptoms vary from small necrotic lesions on the caps of the fruiting bodies to partially deformed fruiting bodies, called stipe blow-out, or totally deformed and undifferentiated masses of mushroom tissue, called dry bubble. The disease symptoms and severity depend on the time point of infection. Small necrotic lesions result from late infections on the fruiting bodies, whereas stipe blow-out and dry bubble are the result of interactions between the pathogen and the host in the casing layer. ECONOMIC IMPORTANCE Lecanicillium fungicola is a devastating pathogen in the mushroom industry and causes significant losses in the commercial production of its main host, Agaricus bisporus. Annual costs for mushroom growers are estimated at 2-4% of total revenue. Reports on the disease originate mainly from North America and Europe. Although China is the main producer of white-button mushrooms in the world, little is known in the international literature about the impact of dry bubble disease in this region. CONTROL The control of L. fungicola relies on strict hygiene and the use of fungicides. Few chemicals can be used for the control of dry bubble because the host is also sensitive to fungicides. Notably, the development of resistance of L. fungicola has been reported against the fungicides that are used to control dry bubble disease. In addition, some of these fungicides may be banned in the near future. USEFUL WEBSITES http://www.mycobank.org; http://www.isms.biz; http://www.cbs.knaw.nl.

Effects of the mushroom-volatile 1-octen-3-ol on dry bubble disease

Dry bubble disease caused by Lecanicillium fungicola is a persistent problem in the cultivation of the white button mushroom (Agaricus bisporus). Because control is hampered by chemicals becoming less effective, new ways to control dry bubble disease are urgently required. 1-Octen-3-ol is a volatile that is produced by A. bisporus and many other fungi. In A. bisporus, it has been implicated in self-inhibition of fruiting body formation while it was shown to inhibit spore germination in ascomycetes. Here, we show that 1-octen-3-ol inhibits germination of L. fungicola and that enhanced levels of 1-octen-3-ol can effectively control the malady. In addition, application of 1-octen-3-ol stimulates growth of bacterial populations in the casing and of Pseudomonas spp. specifically. Pseudomonas spp. and other bacteria have been demonstrated to play part in both the onset of mushroom formation in A. bisporus, as well as the inhibition of L. fungicola spore germination. A potential role of 1-octen-3-ol in the ecology of L. fungicola is discussed.

A detailed analysis of the recombination landscape of the button mushroom Agaricus bisporus var. bisporus

The button mushroom (Agaricus bisporus) is one of the worlds 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.

Quantitative trait locus mapping for bruising sensitivity and cap color of Agaricus bisporus (button mushrooms).

White button mushrooms discolor after mechanical damage of the cap skin. This hampers the development of a mechanical harvest system for the fresh market. To unravel the genetic basis for bruising sensitivity, two haploid populations (single spore cultures) were generated derived from crosses between parental lines differing in discoloration after mechanical damage (bruising sensitivity). The haploids were crossed with different homokaryotic tester lines to generate mushrooms and allow assessment of the bruising sensitivity in different genetic backgrounds. Bruising sensitivity appears to be a polygenic highly heritable trait (H(2): 0.88-0.96) and a significant interaction between genotypes and tester lines and genotypes and flushes was found. Using SNP markers evenly spread over all chromosomes, a very low recombination was found between markers allowing only assignment of QTL for bruising sensitivity to chromosomes and not to sub-regions of chromosomes. The cap color of the two parental lines of population 1 is white and brown respectively. A major QTL for bruising sensitivity was assigned to chromosome 8 in population 1 that also harbors the main determinant for cap color (brown versus white). Splitting offspring in white and non-white mushrooms made minor QTL for bruising sensitivity on other chromosomes (e.g. 3 and 10) more prominent. The one on chromosome 10 explained 31% phenotypic variation of bruising sensitivity in flush 2 in the subpopulations of population 1. The two parental lines of population 2 are both white. Major QTL of bruising sensitivity were detected on chromosome 1 and 2, contributing totally more than 44% variation of the bruising sensitivity in flush 1 and 54% variation of that in flush 2. A considerable consistency was found in QTL for bruising sensitivity in the different populations studied across tester lines and flushes indicating that this study will provide a base for breeding cultivars that are less sensitive for bruising allowing the use of mechanical harvest and automatic postharvest handling for produce for the fresh market. The low recombination between homologous chromosomes, however, underlines the need to introduce a normal recombination pattern found in a subspecies of the button mushroom.

NAD+-dependent glutamate dehydrogenase of the edible mushroom Agaricus bisporus: biochemical and molecular characterization

Abstract The NAD+-dependent glutamate dehydrogenase (NAD-GDH) of Agaricus bisporus, a key enzyme in nitrogen metabolism, was purified to homogeneity. The apparent molecular mass of the native enzyme is 474 kDa comprising four subunits of 116 kDa. The isoelectric point of the enzyme is about 7.0. Km values for ammonium, 2-oxoglutarate, NADH, glutamate and NAD+ were 6.5, 3.5, 0.06, 37.1 and 0.046 mM, respectively. The enzyme is specific for NAD(H). The gene encoding this enzyme (gdhB) was isolated from an A. bisporus H39 recombinant λ phage library. The deduced amino acid sequence specifies a 1029-amino acid protein with a deduced molecular mass of 115,463 Da, which displays a significant degree of similarity with NAD-GDH of Saccharomyces cerevisiae and Neurospora crassa. The ORF is interrupted by fifteen introns. Northern analysis combined with enzyme activity measurements suggest that NAD-GDH from A. bisporus is regulated by the nitrogen source. NAD-GDH levels in mycelium grown on glutamate were higher than NAD-GDH levels in mycelium grown on ammonium as a nitrogen source. Combined with the kinetic parameters, these results suggest a catabolic role for NAD-GDH. However, upon addition of ammonium to the culture transcription of the gene is not repressed as strongly as that of the gene encoding NADP-GDH (gdhA). To date, tetrameric NAD-GDHs with large subunits, and their corresponding genes, have only been isolated from a few species. This enzyme represents the first NAD-GDH of basidiomycete origin to be purified and is the first such enzyme from basidiomycetes whose sequence has been determined.