Process based modelling of plants-fungus interactions explains fairy ring types and dynamics.

Many mushroom-forming fungi can develop circular colonies affecting the vegetation in a phenomenon named fairy rings. Since the nineteenth century, several hypotheses have been proposed to explain how fairy ring fungi form ring-like shapes instead of disks and why they produce negative or positive effects on the surrounding vegetation. In this context, we present a novel process-based mathematical model aimed at reproducing the mycelial spatial configuration of fairy rings and test different literature-supported hypotheses explaining the suppressive and stimulating effects of fungi on plants. Simulations successfully reproduced the shape of fairy rings through the accumulation of fungal self-inhibitory compounds. Moreover, regarding the negative effects of fungi on vegetation, results suggest that fungal-induced soil hydrophobicity is sufficient to reproduce all observed types of fairy rings, while the potential production of phytotoxins is not. In relation to the positive effects of fungi on plants, results show that the release of phytostimulants is needed to reproduce the vegetation patterns associated to some fairy ring types. Model outputs can guide future experiments and field work to corroborate the considered hypotheses and provide more information for further model improvements.

Stage-specific transposon activity in the life cycle of the fairy-ring mushroom Marasmius oreades.

Genetic variability can be generated by different mechanisms, and across the life cycle. Many basidiomycete fungi have an extended somatic stage, during which each cell carries two genetically distinct haploid nuclei (dikaryosis), resulting from fusion of two compatible monokaryotic individuals. Recent findings have revealed remarkable genome stability at the nucleotide level during dikaryotic growth in these organisms, but whether this pattern extends to mutations affecting large genomic regions remains unknown. Furthermore, despite high genome integrity during dikaryosis, basidiomycete populations are not devoid of genetic diversity, begging the question of when this diversity is introduced. Here, we used a Marasmius oreades fairy ring to investigate the rise of large-scale variants during mono- and dikaryosis. By separating the two nuclear genotypes from four fruiting bodies and generating complete genome assemblies, we gained access to investigate genomic changes of any size. We found that during dikaryotic growth in nature the genome stayed intact, but after separating the nucleotypes into monokaryons, a considerable amount of structural variation started to accumulate, driven to large extent by transposons. Transposon insertions were also found in monokaryotic single-meiospore isolates. Hence, we show that genome integrity in basidiomycetes can be interrupted during monokaryosis, leading to genomic rearrangements and increased activity of transposable elements. We suggest that genetic diversification is disproportionate between life cycle stages in mushroom-forming fungi, so that the short-lived monokaryotic growth stage is more prone to genetic changes than the dikaryotic stage.

New disease and first report of marasmioid fungus, Marasmius palmivorus (Sharples), causing white root rot in Arachis hypogaea L.

Groundnut (Arachis hypogaea L.) plants showing distinct symptoms of necrosis of leaves and severe rotting of roots were observed in an agricultural field at Arakkonam, Tamil Nadu, India. The rhizomorphs of the phytopathogenic fungus were obtained from the rotted roots of the diseased plants and were cultured in the laboratory, and based on the morphological characteristics and nucleotide sequencing analysis of ITS and nLSU region, the phytopathogen was identified as Marasmius palmivorus. The isolated fungus produced distinct fruiting bodies (basidiocarps) when grown under the laboratory conditions. The fungus grew as cottony white colonies on the potato dextrose agar medium and were found to contain septate and clamp connections when examined under light microscope. The pathogenicity of the isolated fungus was assessed by inoculating it on healthy groundnut plant under glasshouse conditions; this resulted in the establishment of typical disease symptoms, which confirmed the pathogenicity of the fungus. The fungal pathogen re-isolated from the artificially inoculated plants was used for molecular characterization and confirmed that the organism was M. palmivorus. To the best of our knowledge, this is the first report on the occurrence of M. palmivorus, causing white root rot disease in A. hypogaea L.

1,2,3-Triazine formation mechanism of the fairy chemical 2-azahypoxanthine in the fairy ring-forming fungus Lepista sordida.

2-Azahypoxanthine (AHX) was first isolated from the culture broth of the fungus Lepista sordida as a fairy ring-inducing compound. It has since been found that a large number of plants and mushrooms produce AHX endogenously and that AHX has beneficial effects on plant growth. The AHX molecule has an unusual, nitrogen-rich 1,2,3-triazine moiety of unknown biosynthetic origin. Here, we establish the biosynthetic pathway for AHX formation in L. sordida. Our results reveal that the key nitrogen sources that are responsible for the 1,2,3-triazine formation are reactive nitrogen species (RNS), which are derived from nitric oxide (NO) produced by NO synthase (NOS). Furthermore, RNS are also involved in the biochemical conversion of 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranosyl 5'-monophosphate (AICAR) to AHX-ribotide (AHXR), suggesting that a novel biosynthetic route that produces AHX exists in the fungus. These findings demonstrate a physiological role for NOS in AHX biosynthesis as well as in biosynthesis of other natural products containing a nitrogen-nitrogen bond.

Marasmius oreades agglutinin enhances resistance of Arabidopsis against plant-parasitic nematodes and a herbivorous insect.

BACKGROUND: Plant-parasitic nematodes and herbivorous insects have a significant negative impact on global crop production. A successful approach to protect crops from these pests is the in planta expression of nematotoxic or entomotoxic proteins such as crystal proteins from Bacillus thuringiensis (Bt) or plant lectins. However, the efficacy of this approach is threatened by emergence of resistance in nematode and insect populations to these proteins. To solve this problem, novel nematotoxic and entomotoxic proteins are needed. During the last two decades, several cytoplasmic lectins from mushrooms with nematicidal and insecticidal activity have been characterized. In this study, we tested the potential of Marasmius oreades agglutinin (MOA) to furnish Arabidopsis plants with resistance towards three economically important crop pests: the two plant-parasitic nematodes Heterodera schachtii and Meloidogyne incognita and the herbivorous diamondback moth Plutella xylostella. RESULTS: The expression of MOA does not affect plant growth under axenic conditions which is an essential parameter in the engineering of genetically modified crops. The transgenic Arabidopsis lines showed nearly complete resistance to H. schachtii, in that the number of female and male nematodes per cm root was reduced by 86-91 % and 43-93 % compared to WT, respectively. M. incognita proved to be less susceptible to the MOA protein in that 18-25 % and 26-35 % less galls and nematode egg masses, respectively, were observed in the transgenic lines. Larvae of the herbivorous P. xylostella foraging on MOA-expression lines showed a lower relative mass gain (22-38 %) and survival rate (15-24 %) than those feeding on WT plants. CONCLUSIONS: The results of our in planta experiments reveal a robust nematicidal and insecticidal activity of the fungal lectin MOA against important agricultural pests which may be exploited for crop protection.

The Assembled and Annotated Genome of the Fairy-Ring Fungus Marasmius oreades.

Marasmius oreades is a basidiomycete fungus that grows in so called "fairy rings," which are circular, underground mycelia common in lawns across temperate areas of the world. Fairy rings can be thought of as natural, long-term evolutionary experiments. As each ring has a common origin and expands radially outwards over many years, different sectors will independently accumulate mutations during growth. The genotype can be followed to the next generation, as mushrooms producing the sexual spores are formed seasonally at the edge of the ring. Here, we present new genomic data from 95 single-spore isolates of the species, which we used to construct a genetic linkage map and an updated version of the genome assembly. The 44-Mb assembly was anchored to 11 linkage groups, producing chromosome-length scaffolds. Gene annotation revealed 13,891 genes, 55% of which contained a pfam domain. The repetitive fraction of the genome was 22%, and dominated by retrotransposons and DNA elements of the KDZ and Plavaka groups. The level of assembly contiguity we present is so far rare in mushroom-forming fungi, and we expect studies of genomics, transposons, phylogenetics, and evolution to be facilitated by the data we present here of the iconic fairy-ring mushroom.

Element concentration, daily intake of elements, and health risk indices of wild mushrooms collected from Belgrad Forest and Ilgaz Mountain National Park (Turkey).

The aim of this study was to determine the element content of wild edible and inedible mushroom species (Agaricus campestris, Armillaria ostoyae, Boletus reticulatus, Bondarzewia mesenterica, Bovistella utriformis, Cantharellus cibarius, Marasmius oreades, Megacollybia platyphylla, Meripilus giganteus, Neoboletus erythropus, Panellus stipticus, Phaeotremella foliacea, Pleurotus ostreatus, Podoscypha multizonata, Russula aurea, R. chloroides, R. virescens, T. versicolor, Trametes gibbose, and Trichaptum biforme) collected from the Belgrad Forests and the Ilgaz Mountain National Park. Based on the results of elemental analyses, daily metal intake (DMI) and health risk index (HRI) values of edible mushrooms collected from both localities were also calculated. As, Cd, Cr, Se, P, Hg, Cu, Mn, Fe, Zn, Al, Ca, Mg, and K contents of mushrooms were in the ranges of 0.16-3.45, 0.09-2.4, 0.15-2.34, 0.3-8.13, 0.28-11.44, 14.03-37.81, 3.87-108.57, 6.18-149.77, 11.9-776.1, 5.4-317.4, 7.4-355.2, 15.4-3517.3, 266.0-2500.0, and 628.0-24083.0 mg/kg dry weight, respectively. As a result of the DMI and HRI analyses, Cu concentration of B. utriformis (DMI: 46.53 µg/kg body weight/serving, HRI: 1.16) and Cd concentrations of A. campestris (DMI: 0.49 µg/kg body weight/serving, HRI: 1.36), A. ostoyae (DMI: 1.03 µg/kg body weight/serving, HRI: 2.86), B. utriformis (DMI: 0.52 µg/kg body weight/serving, HRI: 1.44), and P. ostreatus (DMI: 0.45 µg/kg body weight/serving, HRI: 1.24) were found to exceed the legal limits determined by authorities. It was concluded that the species collected from the regions in question should be consumed in a controlled manner.

Napthrene Compounds from Mycelial Fermentation Products of Marasmius berteroi.

The metabolites of the genus Marasmius are diverse, showing good research prospects for finding new bioactive molecules. In order to explore the active metabolites of the fungi Marasmius berteroi, the deep chemical investigation on the bioactive compounds from its cultures was undertaken, which led to the isolation of three new naphthalene compounds dipolynaphthalenes A-B (1,2) and naphthone C (3), as well as 12 known compounds (4-15). Compounds 1, 2, and 4 are dimeric naphthalene compounds. Their structures were elucidated by MS, 1D and 2D NMR spectroscopic data, as well as ECD calculations. Compounds 2-4 and 7 exhibited acetylcholinesterase (AChE) inhibitory activities at the concentration of 50 µg/mL with inhibition ratios of 42.74%, 44.63%, 39.50% and 51.49%, respectively. Compounds 5 and 7,8 showed weak inhibitory activities towards two tumor cell lines, with IC50 of 0.10, 0.076 and 0.058 mM (K562) and 0.13, 0.18, and 0.15 mM (SGC-7901), respectively.

First report of the oil palm disease fungus Marasmius palmivorus from Taiwan causing stem rot disease on native Formosa palm Arenga engleri as new host.

Basidiomata were found on dead stems of wild native Formosa palm (Arenga engleri) in Taiwan. The fungus was identified based on morphology and internal transcribed spacer sequence comparison as the oil palm pathogen Marasmius palmivorus. A pathogenicity test with cultivated mycelium of M. palmivorus positively produced disease symptoms and death of non-wounded Formosa palm tree seedlings under excessive moisture conditions. These results indicate that mycelial inoculum may be more important for pathogenesis than spore inoculum and that the fungus does not require wounds for entry into the plant. Host records in the literature are critically revised. The extended geographical and host distribution indicate a greater risk by M. palmivorus in palm plantations than hitherto anticipated. SIGNIFICANCE AND IMPACT OF THE STUDY: Certain palm species are susceptible to disease caused by Marasmius palmivorus, particularly in oil and coconut palm plantations in tropical countries. Hitherto, there is no published information on the morphology and pathogenicity of the species in Taiwan.

Antioxidant, Anticancer, Antimicrobial, and Antibiofilm Properties of the Culinary-Medicinal Fairy Ring Mushroom, Marasmius oreades (Agaricomycetes).

This study is based on the phenolic composition and the antioxidant, anticancer, antimicrobial, and antibiofilm activities of the edible mushroom Marasmius oreades from Turkey. The phenolic composition of an M. oreades ethanol extract was measured by using the Folin-Ciocalteu method, aluminium chloride colorimetry, and ultraperformance liquid chromatography. The antioxidant activity was evaluated on the basis of DPPH radical scavenging activity. The effect of the M. oreades ethanol extract was also screened in order to determine glutathione-S-transferase, glutathione peroxidase, catalase, and superoxide dismutase enzyme activities. The antimicrobial activity of the mushroom extract was evaluated by using well diffusion and was based on the minimum inhibitory concentration. In addition, the antibiofilm potential of M. oreades was analyzed against Gram-positive and -negative bacteria. Finally, the anticancer effects of the mushroom extract were tested on colon (HT-29) and breast (MCF-7 and MDA-MB-231) cancer cell lines by using the MTT assay. The results revealed that the total amount of phenolics in the ethanol extract of M. oreades was 10.990 ± 0.0007 mg gallic acid equivalent/100 g, and the total amount of flavonoids was 1.139 ± 0.0052 mg quercetin equivalent/100 g. The ultraperformance liquid chromatography results indicated that the M. oreades ethanol extract contained various phenolic compounds: catechin, ferulic, gallic acid, and vanillic acid. The M. oreades ethanol extract scavenged about 80% of DPPH free radicals. It did not show any effect on the glutathione-S-transferase, glutathione peroxidase, and catalase enzyme activities, but its maximal concentration (10 mg/mL) increased superoxide dismutase activity (8%). The ethanol extract of M. oreades showed a moderate anticancer effect on the HT-29, MCF-7, and MDA-MB-231 cell lines. Although the ethanolic extract of the mushroom did not show sufficient antibacterial activity, it presented a strong antibiofilm effect against all studied pathogenic strains at the tested concentrations.

Micetismos: Parte 4: Síndromes tempranos con síntomas complejos / Mushroom poisonings. Part 4: early-onset syndromes with complex symptoms / Intoxicações por cogumelos. Parte 4: Síndromes precoces com sintomas complexos

En esta Parte 4 de la serie de cuatro artículos sobre micetismos se analizan los síndromes que se caracterizan por presentar un período de latencia muy corto, con la aparición de síntomas complejos en menos de 6 horas después de la ingestión de los macromicetos. Se discuten los siguientes micetismos: 1) Toxíndrome muscarínico o colinérgico periférico por especies de Inocybe y Clitocybe. 2) Toxíndrome inmunohemolítico o hemolítico por Paxillus. 3) Toxíndrome neumónico alérgico por Lycoperdon perlatum y por Pholiota nameko. 4) Toxíndrome panterínico o neurotóxico glutaminérgico por compuestos isoxazólicos o síndrome pantherina/muscaria. 5) Toxíndrome coprínico o cardiovascular. 6) Toxíndrome neurotóxico alucinogénico por psilocibina y derivados indólicos. 7) Toxíndrome psicotrópico por estirilpironas y gimnopilinas de Gymnopilus spectabilis o G. junonius. 8) Toxíndrome agudo de rabdomiólisis por Russula subnigricans. 9) Toxíndrome cianogénico por Marasmius oreades. 10) Toxíndrome inmunosupresor por tricotecenos macrocíclicos de Podostroma cornu-damae. 11) Toxíndrome hemolítico debido a ostreolisina de Pleurotus ostreatus y especies relacionadas. Se analizan los síntomas, las toxinas involucradas, los mecanismos de acción, cuando se conocen, y las especies causantes de los micetismos.

This Part 4 of the series of four articles on mushroom poisonings refers to early-onset syndromes, which are characterized by a very short latency period, and the appearance of complex symptoms in less than 6 hours after mushroom ingestion. The following mycetisms are discussed, (1) Peripheral cholinergic, or muscarinic syndrome due to Inocybe and Clitocybe species. (2) Immunohaemolytic or haemolytic syndrome by Paxillus. (3) Allergic pneumonic syndrome due to Lycoperdon perlatum, and Pholiota nameko. (4) Glutaminergic neurotoxic, or pantherinic syndrome by isoxazole compounds or pantherina/muscaria syndrome. (5) Coprinic or cardiovascular syndrome. (6) Hallucinogenic neurotoxic syndrome due to psilocybin and indole derivatives. (7) Psychotropic syndrome by styrylpirones and gymnopilins of Gymnopilus spectabilis or G. junonius. (8) Rhabdomyolysis acute syndrome due to Russula subnigricans. (9) Cyanogenic syndrome by Marasmius oreades. (10) Immunosuppressive syndrome by macrocyclic trichothecenes of Podostroma cornu-damae. (11) Haemolytic syndrome due to ostreolisine of Pleurotus ostreatus, and related species. The symptoms, toxins involved, mechanisms of action, when known, and the species of mushrooms responsible for the mycetisms are analyzed.

Nesta parte 4 da série de quatro artigos sobre intoxicação por cogumelos são analisadas síndromes que se caracterizam por apresentar um período de latência muito breve, com aparecimento de sintomas complexos em menos de 6 horas após a ingestão dos macromicetos. As seguintes intoxicações com cogumelos são discutidas: (1) Toxíndrome muscarínico ou colinérgico periférico por espécies de Inocybe e Clitocybe. (2) Toxíndrome imuno-hemolítica ou hemolítica por Paxillus. (3) Toxíndrome pneumônica alérgica por Lycoperdon perlatum e por Pholiota nameko. (4) Toxíndrome panterínica ou neurotóxica glutaminérgica por compostos isoxazólicos ou síndrome pantherina/muscaria. (5) Toxíndrome coprínica ou cardiovascular (6) Toxíndrome neurotóxico-alucinogênica por psilocibina e derivados indólicos. (7) Toxíndrome psicotrópica por estirilpironas e gimnopilinas de Gymnopilus spectabilis ou G. junonius. (8) Toxíndrome aguda de rabdomiólise por Russula subnigricans. (9) Toxíndrome cianogênica por Marasmius oreades. (10) Toxíndrome imunossupressora por tricotecenos macrocíclicos de Podostroma cornu-damae. (11) Síndrome hemolítica por ostreolisina de Pleurotus ostreatus e espécies relacionadas. São analisados os sintomas, as toxinas envolvidas, os mecanismos de ação, quando conhecidos, e as espécies de cogumelos responsáveis pelas intoxicações.

Maintenance of High Genome Integrity over Vegetative Growth in the Fairy-Ring Mushroom Marasmius oreades.

Most mutations in coding regions of the genome are deleterious, causing selection to favor mechanisms that minimize the mutational load over time [1-5]. DNA replication during cell division is a major source of new mutations. It is therefore important to limit the number of cell divisions between generations, particularly for large and long-lived organisms [6-9]. The germline cells of animals and the slowly dividing cells in plant meristems are adaptations to control the number of mutations that accumulate over generations [9-11]. Fungi lack a separated germline while harboring species with very large and long-lived individuals that appear to maintain highly stable genomes within their mycelia [8, 12, 13]. Here, we studied genomic mutation accumulation in the fairy-ring mushroom Marasmius oreades. We generated a chromosome-level genome assembly using a combination of cutting-edge DNA sequencing technologies and re-sequenced 40 samples originating from six individuals of this fungus. The low number of mutations recovered in the sequencing data suggests the presence of an unknown mechanism that works to maintain extraordinary genome integrity over vegetative growth in M. oreades. The highly structured growth pattern of M. oreades allowed us to estimate the number of cell divisions leading up to each sample [14, 15], and from this data, we infer an incredibly low per mitosis mutation rate (3.8 × 10-12 mutations per site and cell division) as one of several possible explanations for the low number of identified mutations.

Germline Evolution: Sequestered Cells or Immortal Strands?

Mutation accumulation in long-lived fairy-ring mushrooms is orders of magnitude lower than predicted based on per-cell division mutation rates in other organisms. A possible explanation is the maintenance of 'immortal' template-DNA in the active periphery of the fairy ring.

Upscale and characterization of lignin-modifying enzymes from Marasmiellus palmivorus VE111 in a bioreactor under parameter optimization and the effect of inducers.

Testing different pHs, dissolved oxygen concentrations and temperatures, plus the addition of inducers, to optimize ligninolytic enzyme activity, resulted in increased production of laccases, total peroxidases and manganese peroxidases on the order of 2.1-fold, 4.6-fold and 10-fold, respectively; laccases reached 6588 U/mL, total peroxidases reached 3533 U/mL and manganese peroxidase achieved 60 U/mL. Furthermore, an increase in laccase volumetric productivity and in its specific activity was verified. The addition of inducers, such as copper sulphate and manganese sulphate, improved enzymatic activity. In addition, a new previously unidentified laccase isoform was documented by zymography. The present work successfully increased the production of ligninolytic enzymes.

Analgesic effects of Marasmius androsaceus mycelia ethanol extract and possible mechanisms in mice.

Marasmius androsaceus is a medicinal fungus mainly used to treat various forms of pain in China. This study investigated the analgesic effects of an ethanol extract of M. androsaceus (MAE) and its potential molecular mechanisms. Oral administration of MAE (50, 200, and 1000 mg/kg) had significant analgesic effects in an acid-induced writhing test, a formalin test, and a hot-plate test, with effectiveness similar to tramadol (the positive control drug). The autonomic activity test showed that MAE had no harmful effects on the central nervous system in mice. MAE resulted in significantly enhanced levels of noradrenalin and 5-hydroxytryptamine in serum but suppressed both of these neurotransmitters in the hypothalamus after 30 s of hot-plate stimulation. Co-administration with nimodipine (10 mg/kg; a Ca2+ channel blocker) strongly enhanced the analgesic effect in the hot-plate test compared to MAE alone. Moreover, MAE down-regulated the expression of calmodulin-dependent protein kinase II (CaMKII) in the hypothalamus after a 30-s thermal stimulus. These results suggested that the analgesic ability of MAE is related to the regulation of metabolism by monoamine neurotransmitters and Ca2+/CaMKII-mediated signaling, which can potentially aid the development of peripheral neuropathic pain treatments obtained from M. androsaceus.

Analgesic effects of Marasmius androsaceus mycelia ethanol extract and possible mechanisms in mice

Marasmius androsaceus is a medicinal fungus mainly used to treat various forms of pain in China. This study investigated the analgesic effects of an ethanol extract of M. androsaceus (MAE) and its potential molecular mechanisms. Oral administration of MAE (50, 200, and 1000 mg/kg) had significant analgesic effects in an acid-induced writhing test, a formalin test, and a hot-plate test, with effectiveness similar to tramadol (the positive control drug). The autonomic activity test showed that MAE had no harmful effects on the central nervous system in mice. MAE resulted in significantly enhanced levels of noradrenalin and 5-hydroxytryptamine in serum but suppressed both of these neurotransmitters in the hypothalamus after 30 s of hot-plate stimulation. Co-administration with nimodipine (10 mg/kg; a Ca2+ channel blocker) strongly enhanced the analgesic effect in the hot-plate test compared to MAE alone. Moreover, MAE down-regulated the expression of calmodulin-dependent protein kinase II (CaMKII) in the hypothalamus after a 30-s thermal stimulus. These results suggested that the analgesic ability of MAE is related to the regulation of metabolism by monoamine neurotransmitters and Ca2+/CaMKII-mediated signaling, which can potentially aid the development of peripheral neuropathic pain treatments obtained from M. androsaceus.

Antidepressant-like effects of Marasmius androsaceus metabolic exopolysaccharides on chronic unpredictable mild stress-induced rat model.

Marasmius androsaceus (M. androsaceus), a medicinal fungus, has various pharmacological activities including antidepression. The present study investigated the effects of exopolysaccharides obtained during M. androsaceus submerged fermentation in a chronic unpredictable mild stress (CUMS)­induced depression rat model. Similar to fluoxetine (positive drug), 4­week administration of M. androsaceus exopolysaccharides (MEPS) at doses of 6, 30 and 150 mg/kg strongly enhanced bodyweight gain and sucrose consumption, and reduced the immobility time in forced swimming test and tail suspension test in CUMS rats. MEPS resulted in significant enhancement on the levels of noradrenalin, dopamine, 5­hydroxytryptamine (5­HT), and 5­hydroxyindoleacetic acid in the serum and hypothalamus of CUMS rats, as detected by ELISA. Western blotting results revealed that MEPS upregulated the protein expression levels of tyrosine hydroxylase in the hypothalamus of CUMS rats. In conclusion, these results confirmed the antidepressant­like effects of MEPS, and suggested that the monoamine neurotransmitter system is involved in its antidepressive effects in a CUMS rat model. The present study provided evidence for the clinical application of MEPS as an effective agent against depression.

Family of Papain-Like Fungal Chimerolectins with Distinct Ca2+-Dependent Activation Mechanism.

An important function of fungal lectins is to protect their host. Marasmius oreades agglutinin (MOA) is toxic to nematodes and exerts its protective effect through protease activity. Its proteolytic function is associated with a papain-like dimerization domain. The closest homologue of MOA is Polyporus squamosus lectin 1a (PSL1a). Here, we probed PSL1a for catalytic activity and confirmed that it is a calcium-dependent cysteine protease, like MOA. The X-ray crystal structures of PSL1a (1.5 Å) and MOA (1.3 Å) in complex with calcium and the irreversible cysteine protease inhibitor E-64 elucidated the structural basis for their mechanism of action. The comparison with other calcium-dependent proteases (calpains, LapG) reveals a unique metal-dependent activation mechanism relying on a calcium-induced backbone shift and intradimer cooperation. Intriguingly, the enzymes appear to use a tyrosine-gating mechanism instead of pro-peptide processing. A search for potential MOA orthologues suggests the existence of a whole new family of fungal chimerolectins with these unique features.

Purification and Characterization of the Laccase Involved in Dye Decolorization by the White-Rot Fungus Marasmius scorodonius.

Marasmius scorodonius secretes an extracellular laccase in potato dextrose broth, and this enzyme was purified up to 206-fold using (NH4)2SO4 precipitation and a Hi-trap Q Sepharose column. The molecular mass of the purified laccase was estimated to be ~67 kDa by SDS-PAGE. The UV/vis spectrum of the enzyme was nontypical for laccases, and metal content analysis revealed that the enzyme contains 1 mole of Fe and Zn and 2 moles of Cu per mole of protein. The optimal pH for the enzymatic activity was 3.4, 4.0, and 4.6 with 2,2'-azino-bis(3-ethylbenzothazoline-6-sulfonate) (ABTS), guaiacol, and 2,6-dimethoxy phenol as the substrate, respectively. The optimal temperature of the enzyme was 75°C with ABTS as the substrate. The enzyme was stable in the presence of some metal ions such as Ca2+, Cu2+, Ni2+, Mg2++, Mn2+, Ba2+, Co2+, and Zn2+ at a low concentration (1 mM), whereas Fe2+ completely inhibited the enzymatic activity. The enzymatic reaction was strongly inhibited by metal chelators and thiol compounds except for EDTA. This enzyme directly decolorized Congo red, Malachite green, Crystal violet, and Methylene green dyes at various decolorization rates of 63-90%. In the presence of 1-hydroxybenzotriazole as a redox mediator, the decolorization of Reactive orange 16 and Remazol brilliant blue R was also achieved.

Decolourisation Capabilities of Ligninolytic Enzymes Produced by Marasmius cladophyllus UMAS MS8 on Remazol Brilliant Blue R and Other Azo Dyes.

Marasmius cladophyllus was examined for its ability to degradatively decolourise the recalcitrant dye Remazol Brilliant Blue R (RBBR) and screened for the production of ligninolytic enzymes using specific substrates. Monitoring dye decolourisation by the decrease in absorbance ratio of A592/A500 shows that the decolourisation of RBBR dye was associated with the dye degradation. Marasmius cladophyllus produces laccase and lignin peroxidase in glucose minimal liquid medium containing RBBR. Both enzyme activities were increased, with laccase activity recorded 70 times higher reaching up to 390 U L-1 on day 12. Further in vitro RBBR dye decolourisation using the culture medium shows that laccase activity was correlated with the dye decolourisation. Fresh RBBR dye continuously supplemented into the decolourised culture medium was further decolourised much faster in the subsequent round of the RBBR dye decolourisation. In vitro dye decolourisation using the crude laccase not only decolourised 76% of RBBR dye in just 19 hours but also decolourised 54% of Orange G and 33% of Congo red at the same period of time without the use of any exogenous mediator. This rapid dye decolourisation ability of the enzymes produced by M. cladophyllus thus suggested its possible application in the bioremediation of dye containing wastewater.