Cultivation and utility of <Emphasis Type="Italic">Piptoporus betulinus</Emphasis> fruiting bodies as a source of anticancer agents

Piptoporus betulinus is a wood-rotting basidiomycete used in medicine and biotechnology. However, to date, no indoor method for cultivation of this mushroom fruiting bodies has been developed. Here we present the first report of successful production of P. betulinus mature fruiting bodies in artificial conditions. Four P. betulinus strains were isolated from natural habitats and their mycelia were inoculated into birch sawdust substrate supplemented with organic additives. All the strains effectively colonized the medium but only one of them produced fruiting bodies. Moisture and organic supplementation of the substrate significantly determined the fruiting process. The biological efficiency of the P. betulinus PB01 strain cultivated on optimal substrate (moisture and organic substance content of 55 and 65 and 25 or 35 %, respectively) ranged from 12 to 16 %. The mature fruiting bodies reached weight in the range from 50 to 120 g. Anticancer properties of water and ethanol extracts isolated from both cultured and nature-derived fruiting bodies of P. betulinus were examined in human colon adenocarcinoma, human lung carcinoma and human breast cancer cell lines. The studies revealed antiproliferative and antimigrative properties of all the investigated extracts. Nevertheless the most pronounced effects demonstrated the ethanol extracts, obtained from fruiting bodies of cultured P. betulinus. Summarizing, our studies proved that P. betulinus can be induced to fruit in indoor artificial culture and the cultured fruiting bodies can be used as a source of potential anticancer agents. In this respect, they are at least as valuable as those sourced from nature.     

Preservation affects the vegetative growth and fruiting body production of <Emphasis Type="Italic">Cordyceps militaris</Emphasis>

Cordyceps militaris is a model species of Cordyceps fungi, and has been traditionally used as an edible and medicinal fungus due to its richness of bioactive and pharmacological metabolites. The fruiting bodies of this fungus are widely used as healthy food and nutrition supply. In industrial production, fruiting bodies are cultivated on artificial media, but their yield and quality are usually affected by the quality of fungal strains. In this study, the effect of colony growth rate of the fungal strains, fungal age and repeated subculturing on the fungal biomass accumulation was investigated. The results indicated that the fungal biomass was positively correlated with the colony growth rate and not affected by fungal age and the repeated subculturing. The preservation conditions for stock cultures, including choice of cultures, lyophilization, temperature and protective agents were optimized based on the mycelial formation and conidia production in artificial inoculum. The development of fruiting bodies from the fungal strains stored under the optimized preservation conditions were further analyzed to determine the ideal time period of preservation. Results indicated that storing the fungus at 4 °C could maintain the fungal vitality and fruiting body producing capacity for at least 12 months. This study established practical criteria of fungal inoculum for artificial cultivation of fruiting body and provided a simple and efficient preservation method for C. militaris. The results may shed light on preservation for other Cordyceps species and other edible fungi.     

Polluting macrophytes Colombian lake Fúquene used as substrate by edible fungus Pleurotus ostreatus

Invasive aquatic plants from Lake Fúquene (Cundinamarca, Colombia), water hyacinth (Eichhornia crassipes C. Mart.) and Brazilian elodea (Egeria densa Planch.) have been removed mechanically from the lake and can be used for edible mushrooms production. The growth of the oyster mushroom (Pleurotus ostreatus) on these aquatic macrophytes was investigated in order to evaluate the possible use of fruiting bodies and spent biomass in food production for human and animal nutrition, respectively. Treatments included: water hyacinth, Brazilian elodea, sawdust, rice hulls and their combinations, inoculated with P. ostreatus at 3 %. Water hyacinth mixed with sawdust stimulated significantly fruiting bodies production (P = 3.3 × 10^?7) with 71 % biological efficacy, followed by water hyacinth with rice husk (55 %) and elodea with rice husk (48 %), all of these have protein contents between 26 and 47 %. Loss of lignin (0.9–21.6 %), cellulose (3.7–58.3 %) and hemicellulose (1.9–53.8 %) and increment in vitro digestibility (16.7–139.3 %) and reducing sugars (73.4–838.4 %) were observed in most treatments. Treatments spent biomass presented Relative Forage Values (RFV) from 46.1 to 232.4 %. The results demonstrated the fungus degrading ability and its potential use in aquatic macrophytes conversion biomass into digestible ruminant feed as added value to the fruiting bodies production for human nutrition.     

Lactarius deliciosus and Pinus radiata in New Zealand: towards the development of innovative gourmet mushroom orchards

The cultivation of Lactarius deliciosus (saffron milk cap) in New Zealand began in 2002 when fruiting bodies were produced in an Otago plantation of Pinus radiata seedlings artificially mycorrhized by L. deliciosus. In 2007, 42 P. radiata seedlings mycorrhized by L. deliciosus under controlled conditions were planted in a grass field at Plant and Food Research (Lincoln, Canterbury). The effects of pine bark mulch application and initial degree of mycorrhization of seedlings were examined to determine their influence on tree growth, development of mycorrhizae (i.e. their multiplication on the root system and their degree of branching) and fruiting body production. Mulch application increased tree growth significantly over 4 years. High initial mycorrhization slightly stimulated tree growth over 2 years. The initial degree of mycorrhization was positively, but not strongly, related to the persistence and development of L. deliciosus mycorrhizae and rhizomorphs based on root sample analyses 2 years after planting. However, mulching strongly reduced the proportion of highly branched L. deliciosus mycorrhizae compared with poorly ramified ones. A positive correlation was observed between the fruiting of L. deliciosus and the development of mycorrhizae. Mulching delayed the onset of fruiting body production. In 2010, fruiting bodies were produced only from non-mulched trees with eight of these (38 %) producing a total of 12 fruiting bodies. In 2011, 19 non-mulched trees (90 %) and 9 mulched trees (45 %) produced 143 and 47 fruiting bodies, respectively, totalling 190 fruiting bodies. By 2012, 19 non-mulched trees (90 %) and 13 mulched trees (65 %) produced 333 and 236 fruiting bodies, respectively, totalling 569 fruiting bodies (c. 30 kg). This study presents new information on factors influencing the onset of fruiting and the development of yields in a plantation of P. radiata mycorrhized by L. deliciosus. Projected yields as high as c. 300 kg/ha from the third year of production reiterate the feasibility of farming saffron milk cap in P. radiata plantations in New Zealand. Continued monitoring of this site and development of similar trials will provide important knowledge for the optimisation of yields in commercial saffron milk cap orchards.     

Biosynthesis of the carbohydrate moieties of arabinogalactan proteins by membrane-bound β-glucuronosyltransferases from radish primary roots

A membrane fraction from etiolated 6-day-old primary radish roots (Raphanus sativus L. var hortensis) contained β-glucuronosyltransferases (GlcATs) involved in the synthesis of the carbohydrate moieties of arabinogalactan proteins (AGPs). The GlcATs transferred [¹⁴C]GlcA from UDP-[¹⁴C]GlcA on to β-(1 → 3)-galactan as an exogenous acceptor substrate, giving a specific activity of 50–150 pmol min^?1 (mg protein)^?1. The enzyme specimen also catalyzed the transfer of [¹⁴C]GlcA on to an enzymatically modified AGP from mature radish root. Analysis of the transfer products revealed that the transfer of [¹⁴C]GlcA occurred preferentially on to consecutive (1 → 3)-linked β-Gal chains as well as single branched β-(1 → 6)-Gal residues through β-(1 → 6) linkages, producing branched acidic side chains. The enzymes also transferred [¹⁴C]GlcA residues on to several oligosaccharides, such as β-(1 → 6)- and β-(1 → 3)-galactotrioses. A trisaccharide, α-l-Araf-(1 → 3)-β-Gal-(1 → 6)-Gal, was a good acceptor, yielding a branched tetrasaccharide, α-l-Araf-(1 → 3)[β-GlcA-(1 → 6)]-β-Gal-(1 → 6)-Gal. We report the first in vitro assay system for β-GlcATs involved in the AG synthesis as a step toward full characterization and cloning.     

Morphogenesis in Schizophyllum commune. I. Effects of White Light

Reproductive differentiation in the basidiomycete Schizophyllum commune Fr. is initiated by plasmogamy and reciprocal nuclear migration and is terminated by the production of basidiospores. The work reported here has analyzed several factors that affect 2 sequential steps in reproductive differentiation: A) the formation of aggregated masses of cells, and B) the subsequent differentiation of fruiting bodies. The 2 steps are both photosensitive: A) light accelerates the formation of aggregated masses of cells; B) a short exposure of light induces nonaggregated dikaryotic cells to form mature fruiting bodies in the dark.     

Purification an α-galactosidase from Coriolus versicolor with acid-resistant and good degradation ability on raffinose family oligosaccharides

An acid-tolerant α-galactosidase (CVGI) was isolated from the fruiting bodies of Coriolus versicolor with a 229-fold of purification and a specific activity of 398.6 units mg^?1. It was purified to electrophoretic homogeneity by ion exchange chromatography and gel filtration chromatography. The purified enzyme gave a single band corresponding to a molecular mass of 40 kDa in SDS-PAGE and gel filtration. The α-galactosidase was identified by MALDI-TOF-MS and its inner peptides were sequenced by ESI-MS/MS. The optimum temperature and pH of the enzyme were determined as 60 °C and 3.0, respectively. The enzyme was very stable at a temperature range of 4–50 °C and at a pH range of 2–5. Among the metal ions tested, Cu²⁺, Cd²⁺ and Hg²⁺ ions have been shown to partially inhibit the activity of α-galactosidase, while the activity of CVGI was completely inactivated by Ag⁺ ions. N-bromosuccinamide inhibited enzyme activity by 100 %, indicating the importance of tryptophan residue(s) at or near the active site. CVGI had wide substrate specificity (p-nitrophenyl galactoside, melidiose, raffinose and stachyose). After treatment with CVGI, raffinose family oligosaccharide was hydrolyzed effectively to yield galactose and sucrose. The results showed that the general properties of the enzyme offer potential for use of this α-galactosidase in several production processes.     

Bioconversion of α-pinene by a novel cold-adapted fungus <Emphasis Type="Italic">Chrysosporium pannorum</Emphasis>

The psychrotrophic fungus Chrysosporium pannorum A-1 is reported for the first time as a novel biocatalyst for O₂-promoted oxidation of α-pinene. GC–MS analysis indicated that the main products of the reaction were compounds of a high commercial value, verbenol (1) and verbenone (2). Exponentially growing cells (days 2–3) were about twice as active as cells in the late stationary phase in terms of the total concentration of products. The highest yields of 1 and 2 were obtained using three-day and two-day-old mycelia and a medium containing 1.5 and 1 % (v/v) of the substrate, respectively. The optimal time for the bioconversion of α-pinene varied from 1 to 3 days, and depended on the kind of product desired. Most of 1 was produced at a relatively high concentration of 360 mg/L after the first six hours of α-pinene bioconversion [with an average yield of 69 mg/(g _{dry cell} L aqueous phase)]. The oxidative activity of C. pannorum was identified across a wide temperature range of 5–25 °C, 10 °C being the optimum for the production of 1 and 20 °C for the production of 2. Sequential addition of the substrate during 3 days of the biotransformation resulted in a significant increase in 1 and 2 up to 722 and 176 mg/L, respectively, and a 2-fold enhancement of product yield as compared to bioconversion with a single supply of α-pinene. The concentration of total conversion products in the culture medium reached 1.33 g/L [which corresponded product yield of 225 mg/(g _{dry cell} L)]. This represents probably the most promising result reported to date for oxidative biotransformation of α-pinene by a wild-type microorganism.     

Nanofiltration of polysaccharides from Agaricus subrufescens

A simplified submerged airlift cultivation was established for the production of biomass from Agaricus subrufescens. In this work, soluble polysaccharides extracted from fungal mycelium, fruiting bodies, and the residual culture media were concentrated by nanofiltration. Total and high molar mass polysaccharides and soluble solids were determined in the concentrate for the three extracts. Additionally, the permeate flow, the influences of temperature and pressure, and the resistance to the permeate flow during filtration were also evaluated. A yield of 5.5 g/L of biomass with 35 % glucose conversion was obtained when 0.5 g/L of initial inoculum was employed. Average specific speed of growth was 0.4/day, with biomass productivity of about 0.76 g/(L day). Nanofiltration has yielded polysaccharide increases of 85, 82, and 92 % in the extracts from fruiting bodies, mycelium, and liquid media, respectively. A reduction in the permeate flow was observed during filtration, and it was compensated by higher pressures and temperatures. The higher resistance to the permeate flux was caused by polarization due to concentration (polarized gel layer), reaching values of 88 % for the culture media. Maximal resistance caused by the membrane reached values of 40 % for the extract from the fruiting bodies. On the other hand, resistance caused by fouling was responsible for less than 3.5 %. In conclusion, nanofiltration is efficient to concentrate these functional compounds extracted from A. subrufescens and can, therefore, be applied in different biotechnological areas.     

Antioxidant components of <Emphasis Type="Italic">Laetiporus sulphureus</Emphasis> (Bull.: Fr.) Murr. fruit bodies

Antioxidant activity of fruit bodies of Laetiporus sulphureus (Bull.: Fr.) Murr. (Polyporales) obtained by the natural plantation growing method in Pribaikal’e (Irkutsk region) has been studied. It was determined that the ethyl acetate fraction of L. sulphureus, which was chromatographically separated into seven compounds identified as quercetin, kaempferol, (+)-catechin, p-coumaric, gallic, caffeic, and chlorogenic acids was characterized with more expressed antioxidant activity. All compounds were extracted from this basidiomycete species for the first time. The quantitative amount of the substances isolated from L. sulphureus was determined by HPLC. It was found that antioxidant activity of preparations obtained from L. sulphureus is conditioned by phenolic compounds.     

Relationships Between Single Nucleotide Polymorphisms of the H-FABP Gene and Slaughter and Meat Quality Traits in Chicken

Using PCR-SSCP with five primer pairs, we detected six single nucleotide polymorphisms of the H-FABP gene: 332G → A, 534G → A, 783C → T, 835C → T, 1198T → C, and 2329C → T. Chi-square results showed significant differences (P < 0.05) in genotype frequency among breeds in Fragment 1 and extremely significant differences (P < 0.01) in Fragments 2–4. We found a significant association between Fragment 2 genotype and muscle fiber number, Arg and Thr (P < 0.05); between Fragment 3 genotype and living weight, carcass weight, breast muscle weight, abdominal fat weight, and abdominal fat percentage (P < 0.05); between Fragment 4 genotype and Thr, Phe, and inosinic acid (P < 0.05). It was concluded that H-FABP was the major gene influencing slaughter performance and meat quality or was linked with the major gene in these strains and that the C783T mutation could be used as a candidate molecular genetic marker for breeding selection. The combination M1C2–B2B2–D1D1 is an ideal model for breeding in these strains because it can improve slaughter and meat quality traits.     

Infection and colonization of tissues of the aphid Myzus persicae and cassava mealybug Phenacoccus manihoti by the fungus Beauveria bassiana

Histopathogenesis of living insects of Myzus persicae Sulzer (Hemiptera: Aphididae) and Phenacoccus manihoti Matile‐Ferrero (Hemiptera: Pseudococcidae) by Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) was monitored from penetration through insect death. Important events in aphids included fungal penetration of the integument of the less-resistant leg intersegmental membrane and invasion of natural openings, formation of hyphal bodies in live aphids by three days post-inoculation (PI), and extensive hyphal colonization of the two leg segments closest to the insect body at death of the aphids. Confocal microscopy of green fluorescent protein-labeled B. bassiana in live mealybugs indicated the fungus penetrated the host through the legs and mouthparts. The fungus was scarce in live mealybugs at 1–5 days PI, formed hyphal bodies by six days PI, and growth was limited to parts of dead hosts at 6–7 days PI. In dead mealybugs, hyphal bodies were near solid tissue. Blastospores were in the hemolymph.     

Determination of volatile organic compounds in the stinkhorn fungus Pseudocolus fusiformis in different stages of fruiting body formation

A stinkhorn fungus was collected from the mountainous area of Yoshida campus, Yamaguchi University, Japan. Morphological characterization and similarity of large subunit ribosomal DNA sequences identified the fungus as Pseudocolus fusiformis. MonoTrap™ was combined with gas chromatography-mass spectrometry (GC-MS) to identify volatile organic compounds (VOCs) emitted from the fungus harvested at different stages of maturity. The main VOCs emitted from the mature fruiting body were 3-methyl-butanol, 4-methyl-phenol, and dimethyl tetrasulfide, while none of these compounds were detected in the egg-shaped state. Volatile sulfur-containing compounds, including dimethyl disulfide, trisulfide and tetrasulfide, which are commonly detected in stinkhorn fungi and truffles, were also emitted from this fungus. Furthermore, results elucidated that most VOCs occurred in the mature stage of Ps. fusiformis (fruiting body with arms fuse). This is the first study reporting VOC production of Ps. fusiformis.     

Physiology of Lichtheimia ramosa obtained by solid-state bioprocess using fruit wastes as substrate

Due to the amount of nutrients available in the agroindustrial wastes, these can be converted into high added-value products by the action of microorganisms in solid-state bioprocesses. The aim of this work was to evaluate the growth physiology and lipase production of the fungus Lichtheimia ramosa using the following Brazilian savannah fruit wastes as substrates: bocaiuva (Acrocomia aculeata), pequi (Caryocar brasiliense), guavira (Campomanesia pubescens), araticum (Annona crassiflora) and seriguela (Spondias purpurea). These residues were triturated, homogenized, adjusted to pH 5.0 and 60 % moisture, sterilized and packaged in plastic tray-type bioreactors before inoculation with 10 % (w/v) of L. ramosa pre-culture medium. The cultivations were conducted in a bacteriological incubator at 30 °C for 40 days. Samples were taken every 5 days and fungi and bacteria contents, proximate composition and lipase activity were evaluated. The maximum fungal counting was observed between 25 and 35 days. L. ramosa reached the stationary phase next to 40 days in all substrates. Mesophilic and psicrophilic aerobic bacteria were not detected. Protein enrichment was obtained for all media, being superior in seriguela residues (391.66 %), followed by pequi (160.04 %), araticum (143.31 %), guavira (102.42 %), and bocaiuva (67.88 %). Lipase production was observed in all cultivated media, except in pequi residues that showed decreasing lipase activity. The higher production was observed in guavira (1.12 U/g) followed by araticum (0.58 U/g), seriguela (0.41 U/g) and bocaiuva (0.21 U/g) waste substrates. It was concluded that the studied fruit wastes have been successfully utilized as substrates for protein enrichment and lipase production with L. ramosa.     

Accumulation and decay dynamics of coarse woody debris in a Japanese old-growth subalpine coniferous forest

Far less is known about the coarse woody debris (CWD) stock and decay process in temperate Asia compared with that in boreal and temperate Europe and North America. We estimated coniferous CWD stock (logs and snags), decay rate and process, and fungal species responsible for the decay process in a Japanese subalpine coniferous forest. The CWD mass was 42.4 Mg ha^?1, which was the greatest among the previous data recorded in temperate Asia. The decay rate calculated using the annual input of CWD divided by CWD accumulation was 0.036 year^?1, whereas the decay rate when measured chronosequentially was 0.020–0.023 year^?1. The decay process was divided into two phases characterized by different dominant organic chemical constituents. In the first phase, both acid-unhydrolyzable residue and holocellulose decayed simultaneously, suggestive of the white-rot process. In the second phase, holocellulose was selectively decomposed and AUR accumulated, suggestive of the brown-rot process. Nutrients (N, P, K, Na, Mg, and Ca) were mineralized in the first phase but immobilized in the second phase. The fruiting bodies of 26 taxa of fungi were recorded as occurring on CWD in the study area. Trichaptum abietinum and T. fuscoviolaceum, which dominated in the first phase and are known as white-rot fungi, were assumed to be the main decomposers of lignocellulose in the first phase. Although no known strong wood decomposers dominated the second phase, Laetiporus sulphureus and Oligoporus caesius, known as brown-rot fungi, were expected to participate in the selective decomposition of holocellulose in the second phase.     

Agrobacterium tumefaciens-mediated transformation of SOD gene to Trichoderma harzianum

Trichoderma harzianum is a soil-borne filamentous fungus that exhibits biological control properties because it parasitizes a large variety of phytopathogenic fungi. In this study the SOD gene was successfully transferred into the bio-control fungus Trichoderma harzianum with an efficiency of 60–110 transformants per 10⁷ spores by using Agrobacterium tumefaciens-mediated transformation. Putative transformants were analyzed to test the transformation by the southern blot. Antifungal activities of the transformants were examined under abiotic stresses. The transformants were exposed to 40°C for three days and 2 mol/l NaCl at 27°C for 5–10 days to assay antifungal activities with Sclerotinia sclerotiorum. The inhibition rates of the transformants, comparing to Trichoderma harzianum with no SOD gene transferred, were respectively 83.96% after 40°C and 60.13% after 2 mol/l NaCl. The results showed that the SOD transformants had significantly higher resistance to heat and salt stress.     

Protein expression during Flammulina velutipes fruiting body formation

Formation of the Flammulina velutipes fruiting body can be induced by lowering the ambient temperature (first treatment) in complete darkness. Fruiting bodies formed under these conditions elongate without pileus formation (pinhead fruiting body), suggesting that they cannot mature in complete darkness. However, after light treatment of the pinhead fruiting body (second treatment), a pileus develops immediately, and the stipe also thickens and becomes increasingly pigmented. The apical region swells as a result of cell division starting 2 days after light treatment, the pileus–stipe junction fracture and hymenium primordia form on day 4, and gills appear at day 6. Pf1 and Pf3 are specifically expressed after exposure to low temperature without light. The cell wall-associated protein [pileus-specific hydrophobin-like protein (PSH)] is specifically induced in the pileus, but not in the stipe, following the second light treatment to the pinhead fruiting body. These results suggest that Pf1 and Pf3 would be involved in fruiting body induction and that PSH would be involved in pileus formation. These phenomena will aid further histological and molecular biological investigations into the mechanisms behind fruiting body development in F. velutipes.