Trichoderma songyi sp. nov., a new species associated with the pine mushroom (Tricholoma matsutake)

A new species, Trichoderma songyi, was found to be associated with the pine mushroom (Tricholoma matsutake) in Korea. This species was isolated from three different substrates: Tricholoma matsutake basidiomata, as well as roots of Pinus densiflora and soil in the fairy ring. Based on its molecular and phenotypic characteristics, we demonstrate that Trichoderma songyi is unique and distinguishable from closely related species. We performed phylogenetic analyses based on two molecular markers, the genes for both translation elongation factor 1-alpha and the second largest subunit of RNA polymerase II. Phylogenetic analyses showed that Trichoderma songyi is closely related to Trichoderma koningii aggregate and Trichoderma caerulescens. Morphologically, Trichoderma songyi can be distinguished from these closely related taxa by its growth rates, colony morphology on PDA in darkness, and coconut-like odour. Due to the economic importance of the pine mushroom, the relationship between Trichoderma songyi and Tricholoma matsutake should be studied further.     

Isolation by landscape in populations of a prized edible mushroom Tricholoma matsutake

Tricholoma matsutake, a wild edible ectomycorrhizal mushroom, is revered for its distinguished flavor and iconic significance. Here, we test for landscape effects on T. matsutake gene flow and population structure in the Eastern Himalayas. Using single-nucleotide polymorphic (SNP) DNA markers, isolation by distance patterns were tested on eight populations within and between watersheds. We find that high, treeless ridgelines are effective barriers to gene flow, even at distances less than 65 km, whereas populations located within watersheds are structured at greater distances. Mantel tests demonstrated a significant positive correlation between F _st and a “landscape distance” measured as the shortest distance between population pairs below treeline r = 0.574, P = 0.002, whereas strict euclidian distances do not correlate. AMOVA analysis revealed significant partitioning with 91% of the genetic variance found within populations and 7% found between watersheds, indicative of sexually recombining populations with limited gene flow between watersheds. We show that landscape is an important determinant of air-dispersed ectomycorrhizal species population structure in heterogeneous landscapes.     

Purification, chemical characterization and radical scavenging activities of alkali-extracted polysaccharide fractions isolated from the fruit bodies of Tricholoma matsutake

Tricholoma matsutake, a high-class edible mushroom in China, has been regarded as famous foods and biopharmaceutical materials with a great deal of interest. In the previous investigations, researchers believe the water-soluble polysaccharide β-glucan is the major active component of T. matsutake, which displays various biological activities. In the present study, two novel alkali-extracted polysaccharide fractions, TM-APS-1 and TM-APS-2, were isolated from the fruit bodies of T. matsutake by DEAE-cellulose and Sepharose CL-6B columns on ÄTKA explorer chromatography system. Their chemical and physical characteristics and radical scavenging capacity were valuated, including chemical methods, GC, HPLC, scavenging activity against DPPH radicals, superoxide radicals, hydroxyl radicals, and chelating ability. The results showed that TM-APS-1 and TM-APS-2 exhibited significantly antioxidant activity at a concentration-dependent manner. The alkali-extracted polysaccharide fractions from T. matsutake can be developed to be novel functional food or pharmaceutical products with antioxidant effects.     

In vitro ectomycorrhization of Tricholoma matsutake strains is differentially affected by soil type

The Japanese delicacy Tricholoma matsutake has been conducted in vitro ectomycorrhizal syntheses for more than 20 y. The development of its ectomycorrhizal structures varies among experimental systems. Here, we examined the effects of soil-fungus interactions on the early stage of in vitro T. matsutake ectomycorrhization. Axenic Pinus densiflora seedlings were transplanted into autoclaved natural inorganic soil, inoculated with the cultured mycelium of T. matsutake, and incubated for 90 d in vitro. Both soil type and fungal strain significantly affected host plant growth; host plant growth and mycorrhization levels significantly differed among soil type/fungal strain combinations. Therefore, the selection of T. matsutake strains for optimal mycorrhization must take into account such fungal and soil properties.     

The extraction process optimization and physicochemical properties of polysaccharides from the roots of Euphorbia fischeriana

Response surface methodology (RSM) was used to determine the optimum extraction conditions for polysaccharides (EFP) from the roots of Euphorbia fischeriana. A Box-Behnken design (BBD) with four independent variables was investigated, such as extraction temperature (°C), water/solid ratio, extraction number (n), and extraction time (h). The results indicated optimum extraction conditions were extraction temperature of 97 °C, water/solid ratio of 9:1, extraction number of 2 and extraction time of 2.4 h, respectively. Under these conditions, the experimental value was 24.6 ± 0.62, which was well in close agreement with value predicted by the model. The preliminary chemical analysis of EFP revealed the EFP contained 25.43% polysaccharides, 20.42% uronic acids, 2.54% sulfate radical and 23.41% proteins. And the neutral polysaccharides were mainly composed of glucose, arabinose, rhamnose, galactose, xylose, mannose in the ratio of 21:8:5:3:1:1.     

Root endophyte interaction between ectomycorrhizal basidiomycete Tricholoma matsutake and arbuscular mycorrhizal tree Cedrela odorata, allowing in vitro synthesis of rhizospheric “shiro”

The ectomycorrhizal basidiomycete Tricholoma matsutake associates with members of the Pinaceae such as Pinus densiflora (red pine), forming a rhizospheric colony or “shiro,” which produces the prized “matsutake” mushroom. We investigated whether the host specificity of T. matsutake to conifers is innately determined using somatic plants of Cedrela odorata, a tropical broad-leaved tree (Meliaceae) that naturally harbors arbuscular mycorrhizal fungi. We found that T. matsutake could form in vitro shiro with C. odorata 140 days after inoculation, as with P. densiflora. The shiro was typically aromatic like that of P. densiflora. However, this was a root endophytic interaction unlike the mycorrhizal association with P. densiflora. Infected plants had epidermal tissues and thick exodermal tissues outside the inner cortex. The mycelial sheath surrounded the outside of the epidermis, and the hyphae penetrated into intra- and intercellular spaces, often forming hyphal bundles or a pseudoparenchymatous organization. However, the hyphae grew only in the direction of vascular bundles and did not form Hartig nets. Tricholoma fulvocastaneum or “false matsutake” naturally associates with Fagaceae and was also able to associate with C. odorata as a root endophyte. With T. matsutake, C. odorata generated a number of roots and showed greatly enhanced vigor, while with T. fulvocastaneum, it generated a smaller number of roots and showed somewhat lesser vigor. We argue that the host–plant specificity of ectomycorrhizal matsutake is not innately determined, and that somatic arbuscular mycorrhizal plants have a great potential to form mutualistic relationships with ectomycorrhizal fungi.     

In vitro ectomycorrhizal specificity between the Asian red pine Pinus densiflora and Tricholoma matsutake and allied species from worldwide Pinaceae and Fagaceae forests

Tricholoma matsutake produces commercially valuable, yet uncultivable, mushrooms (matsutake) in association with pines in the Far East and Scandinavia and with both pines and oaks in the foothills of Tibet. Other matsutake mushrooms, such as Tricholoma anatolicum from the Mediterranean regions and Tricholoma magnivelare and Tricholoma sp. from the North Pacific Coast area of Canada and North America as well as Mexico, respectively, are associated with pines or oaks in their natural habitats. Tricholoma bakamatsutake and Tricholoma fulvocastaneum from Asia produce moderately valuable matsutake mushrooms and are solely associated with Fagaceae in nature. In this study, we demonstrate for the first time that matsutake mushrooms from Scandinavia, Mediterranean regions, North America, and Tibet form ectomycorrhizae with Pinus densiflora similar to the Far East T. matsutake. In general, worldwide T. matsutake and the symbionts of Pinaceae colonize the rhizospheres of P. densiflora as well as T. matsutake isolated from the host plant. However, T. fulvocastaneum and T. bakamatsutake formed a discontinuous Hartig net and no Hartig net, respectively, and colonized to a lesser extent as compared to T. matsutake. The data suggest that conifer-associated matsutake mushrooms in their native habitat will associate symbiotically with the Asian red pine.     

Enzymatic characterization of an extracellular glucoamylase from Tricholoma matsutake and its cloning and secretory expression in Pichia pastoris

ABSTRACT

A glucoamylase from the ectomycorrhizal fungus Tricholoma matsutake (TmGLA) was purified 33.2-fold to homogeneity as a single monomeric glycoprotein with a molecular mass of 63.9 kDa. Maximum activity was observed at 60°C and pH 5.0. The enzyme is active down to 50°C and in the pH range of 4.0–6.0, and its activity is strongly inhibited by Ag⁺. It degrades α-1,4- and α-1,6-glycosidic linkages in various polysaccharides. Its gene (TmGlu1) was cloned using information from the enzyme’s internal amino acid sequences and the whole genome sequence of T. matsutake NBRC 30605. The deduced amino acid sequence showed clear homology with those of GH family 15 proteins. Pichia pastoris transformed with TmGlu1 secreted the active enzyme in a glycosylated form, and its characteristics were the same as the native enzyme.     

Enzyme assisted extraction of polysaccharides from the fruit of Cornus officinalis

Process of enzyme assisted extraction (EAE) of polysaccharides from Cornus officinalis was optimized by response surface methodology (RSM). The influence of four different factors on the yield of C. officinalis polysaccharides (COP) was studied. Results showed that the optimal conditions were compound enzyme amount of 2.15%, extraction pH of 4.2, extraction temperature of 55 °C and extraction time of 97 min. Under these conditions, the COP yield was 9.29 ± 0.31%, which was well in agreement with the value predicted by the model. The three methods, EAE, hot water extraction (HWE), ultrasound-assisted extraction (UAE) for extracting COP by RSM were further compared. Results showed that EAE had the largest yield of polysaccharides with lower equipment cost.     

Physiological variation among Tricholoma matsutake isolates generated from basidiospores obtained from one basidioma

Matsutake (Tricholoma matsutake) is a commercially valuable edible ectomycorrhizal mushroom. The physiological traits of T. matsutake have been previously assessed using mycelial isolates isolated from basidiomata; however, few studies have focused on basidiospores. Here, we report that sibling T. matsutake isolates generated from basidiospores on a single basidioma show distinct physiological variation. We first established 145 isolates of T. matsutake on modified Norkrans' C (MNC) agar medium and found that their radial growth varied significantly. The mycelial biomasses of nine isolates with different growth rates were reduced on low-carbon and low-nitrogen MNC media. However, the colony diam of one isolate was significantly elevated on low-carbon medium, and the colony diam of two isolates were significantly elevated on low-nitrogen medium. In co-cultures of two or three isolates, commensal and amensal interactions were observed. The physiological variation induced by low carbon and nitrogen levels and the mycelial interactions between sibling isolates imply mechanisms for the genetic and functional characteristics of mycelia of T. matsutake.     

The (oxalato)aluminate complex as an antimicrobial substance protecting the “shiro” of Tricholoma matsutake from soil micro-organisms

Tricholoma matsutake, a basidiomycete, forms ectomycorrhizas with Pinus densiflora as the host tree. Its fruiting body, “matsutake” in Japanese, is an edible and highly prized mushroom, and it grows in a circle called a fairy ring. Beneath the fairy ring of T. matsutake, a whitish mycelium-soil aggregated zone, called “shiro” in Japanese, develops. The front of the shiro, an active mycorrhizal zone, functions to gather nutrients from the soil and roots to nourish the fairy ring. Bacteria and sporulating fungi decrease from the shiro front, whereas they increase inside and outside the shiro front. Ohara demonstrated that the shiro front exhibited antimicrobial activity, but the antimicrobial substance has remained unidentified for 50 years. We have identified the antimicrobial substance as the (oxalato)aluminate complex, known as a reaction product of oxalic acid and aluminum phosphate to release soluble phosphorus. The complex protects the shiro from micro-organisms, and contributes to its development.     

The ectomycorrhizal fungus Tricholoma matsutake biosynthesizes benzoic acid and benzaldehyde independently

Metabolism of benzoic acids and benzaldehydes are crucial to produce hormones, defense compounds and attractants for pollinators in plants. Tricholoma matsutake contains benzoic acid and benzaldehyde, but their roles have not been fully studied. First we conducted tracer experiments to gain insight into benzoic acid and benzaldehyde biosynthesis in T. matsutake. ¹³C and ²H were incorporated into benzoic acid from uniformly ¹³C- and ²H- labelled l-phenylalanine and (E)-cinnamate 1?d after supplementation of the precursors without any substitution of ¹³C and ²H. In contrast, no ¹³C and ²H were incorporated into benzaldehyde from these precursors 10?d after the supplementation. The results indicate that T. matsutake has a metabolic pathway to biosynthesize benzoic acid from l-phenylalanine and (E)-cinnamate in which benzaldehyde is not a metabolic intermediate. However, 30?d after the supplementation of ¹³C- and ²H- labelled l-phenylalanine, ¹³C and ²H were incorporated into all the carbon and hydrogen atoms of benzaldehyde. In addition, ²H was not incorporated into benzaldehyde from exogenously supplemented ²H-labelled benzoic acid. This result indicates that T. matsutake has a metabolic pathway to biosynthesize benzaldehyde from l-phenylalanine in which benzoic acid is not an intermediate. Taken together, our results strongly suggest that T. matsutake biosynthesizes benzoic acid and benzaldehyde in separate pathways.     

Growth stimulation of a Shiro-like,mycorrhiza forming,mycelium of <Emphasis Type="Italic">Tricholoma matsutake</Emphasis> on solid substrates by non-ionic surfactants or vegetable oil

The incorporation of Tweens (1 %, 2 %, 5 %) or olive oil (1 %, 2 %) in soil or in soil-containing substrate strongly stimulated mycelial growth of the edible ectomycorrhizal mushroom Tricholoma matsutake (Matsutake) after 1 or 3 months, respectively. The growth responses to Tween 40 and Tween 80 were dose-dependent. Fungal biomass increased up to 15-fold as a result of olive oil incorporation. After 4 months of Matsutake/pine co-culture in the presence of olive oil (2 %), compact aggregates of substrate, hyphae, and surface-colonized roots were observed, recalling in some ways the mycelial mat structure of Matsutake in the field, i.e. Shiro. Olive oil did not prevent formation of well-developed Hartig net palmettis although those seemed rather less abundant than without oil addition. The incorporation of Tween 80 or olive oil (2 %) into nutrient agar induced the proliferation of peripheral hydrophilic-like hyphae penetrating the medium. Tricholoma matsutake growth stimulation, possibly related to the presence of fatty acids in surfactants and oil, could be a consequence of the higher hydrophilicity of treated hyphae, or of enhanced lytic enzyme excretion and activity. Parameters such as adjuvant type, concentration, and growth conditions will be further optimised to formulate culture substrates adapted to the co-culture of T. matsutake and its host plants.     

Detection of β-glucosidase as saprotrophic ability from an ectomycorrhizal mushroom, Tricholoma matsutake

We investigated extracellular carbohydrase production in the medium of an ectomycorrhizal fungus, Tricholoma matsutake, to reveal its ability to utilize carbohydrates such as starch as a growth substrate and to survey the saprotrophic aspects. We found β-glucosidase activity in the static culture filtrate of this fungus. The β-glucosidase was purified and characterized. The purified enzyme was obtained from about 2.1 l static culture filtrate, with 9.0% recovery, and showed a single protein band on SDS-PAGE. Molecular mass was about 160 kDa. The enzyme was most active around 60°C and pH 5.0, and stable over a pH of 4.0–8.0 for 30 min at 37°C. The purified enzyme was activated by the presence of Ca²⁺ and Mn²⁺ ions (about 2–3 times that of the control). The enzyme readily hydrolyzed oligosaccharides having a β-1,4-glucosidic linkage such as cellobiose and cellotriose. However, it did not hydrolyze polysaccharides such as avicel and CM-cellulose or oligosaccharides having an α-glucosidic linkage. Moreover, cellotriose was hydrolyzed by the enzyme for various durations, and the resultant products were analyzed by TLC. We concluded that the enzyme from T. matsutake seems to be a β-glucosidase because cellotriose with a β-1,4-glucosidic linkage decomposed to glucose during the enzyme reaction.     

Dynamic evolution of eukaryotic mitochondrial and nuclear genomes: a case study in the gourmet pine mushroom Tricholoma matsutake

Fungi, as eukaryotic organisms, contain two genomes, the mitochondrial genome and the nuclear genome, in their cells. How the two genomes evolve and correlate to each other is debated. Herein, taking the gourmet pine mushroom Tricholoma matsutake as an example, we performed comparative mitogenomic analysis using samples collected from diverse locations and compared the evolution of the two genomes. The T. matsutake mitogenome encodes 49 genes and is rich of repetitive and non-coding DNAs. Six genes were invaded by up to 11 group I introns, with one cox1 intron cox1P372 showing presence/absence dynamics among different samples. Bioinformatic analyses suggested limited or no evidence of mitochondrial heteroplasmy. Interestingly, hundreds of mitochondrial DNA fragments were found in the nuclear genome, with several larger than 500 nt confirmed by PCR assays and read count comparisons, indicating clear evidence of transfer of mitochondrial DNA into the nuclear genome. Nuclear DNA of T. matsutake showed a higher mutation rate than mitochondrial DNA. Furthermore, we found evidence of incongruence between phylogenetic trees derived from mitogenome and nuclear DNA sequences. Together, our results reveal the dynamic genome evolution of the gourmet pine mushroom.     

Forest age correlates with fine-scale spatial structure of Matsutake mycorrhizas

Examining the fine-scale spatial structure of fungal populations can tell us much about how individual species reproduce and disperse throughout natural landscapes. Here we study the fine-scale genetic structure of Tricholoma matsutake, a prized edible and medicinal mushroom, by systematic sampling of mycorrhizas within fairy rings in 50-y-old and old-growth forests in two villages. Using single nucleotide polymorphism DNA markers we show that mycorrhizas in both forest age classes in both villages showed high levels of genotypic diversity, consistent with a reproductive life history predominated by outcrossing via basidiospore dispersal. Both the percentage of polymorphic loci within fairy rings, as well as genotype diversity were higher in old-growth compared to 50-y-old forests. Fifty-year-old forests showed significant spatial autocorrelation between pairs of mycorrhizas up to 42 m, and a pattern consistent isolation-by-distance structure. Spatial patterns in old-growth forests were random. Furthermore, AMOVA analysis indicates that 11 % of molecular variance in 50-y-old forests is partitioned between villages, whereas no significant variance is partitioned between villages in old-growth forests. We conclude that populations of T. matsutake in 50-y-old forests are the result of a founder effect maintained by local inoculation sources. This pattern attenuates as forests age and accumulate inocula from more distance sources. We speculate on how genetic mosaicism within T. matsutake fairy rings may structure populations within a chronosequence. Finally, we discuss how population spatial dynamics and dispersal strategy in T. matsutake contrast with other ectomycorrhizal species.     

Response surface method was used to optimize the ultrasonic assisted extraction of flavonoids from Crinum asiaticum

In this study, ultrasound-assisted extraction of flavonoid from Crinum asiaticum was studied through response surface methodology (RSM) to gain the best extraction process of flavonoid and enhance the extraction rate of flavonoid. In the following RSM experiment, we selected the corresponding data of every factor as the center point through the single-factor experiments, then the experimental data was subjected to multiple regression analysis. According to the statistical analysis results, the results were consistent with the polynomial regression model, the determination coefficient (R2) was 0.9769. The best conditions for maximum flavonoid yield were 60% ethanol concentration, 64 °C for extraction temperature, 1:28 (v/w) solid-to-liquid ratio with extraction time for 47 min. The best response of flavonoid yield was 1.63972%. The predicted results for best reaction conditions were in good agreement with experiment values. Ultrasound-assisted extraction method can enhance the extraction rate of flavonoid significantly. It is a powerful tool to extract of important phytochemicals from nature plant.     

Bioactive properties of hydroalcoholic extract from Origanum onites L. as affected by glycerol incorporation

In the current study, the effect of glycerol -as a green solvent- addition into solvent mixture (50:50 ethanol:distilled water), on some biofunctional properties of Origanum onites L. was investigated. Response surface methodology (RSM) was used to detect the optimum conditions for the extraction process. Three variables namely glycerol concentration (X₁: 1–9 g), extraction temperature (X₂: 25–75 °C) and time (X₃:10–30 min) were selected and also total phenolic content, total flavonoid content, antioxidant capacity and antiradical activity of O. onites extracts were determined. Analysis of variance (ANOVA) showed that glycerol incorporation significantly increased the total phenolic content and antioxidant activity (p < 0.05) of the samples. Maximum levels to obtain the highest bioactive properties (highest total phenolic content and antioxidant activity) were determined as to be 9 g of glycerol addition for the extraction conditions as 45.4 °C and 75 min. This study reports the effect of glycerol on bioactive properties of O. onites and suggests that glycerol can be used to produce hydroalcoholic extracts having higher bioactivity from Origanum genus.     

Application of Factorial Design and Box–Behnken Matrix in the Optimization of a Magnetic Nanoparticles Procedure for Copper Determination in Water and Biological Samples

This study describes the development by response surface methodology (RSM) of a procedure for copper determination by inductively coupled plasma optical emission spectrometry (ICP-OES) in water and biological samples after extraction by magnetic nanoparticles. Four variables such as, pH of solution, amount of extractant, amount of nanoparticles, and time were regarded as factors in the optimization study. Results of the two-level full factorial design (2⁴) based on an analysis of variance demonstrated that only the pH, amount of extractant (E), and amount of nanoparticles (N) were statistically significant. Optimal conditions for the extraction of copper samples were obtained by using Box–Behnken design. Optimum conditions were 5.1, 7.2 mg, and 9.6 mg, for pH of solution, amount of nanoparticles, and amount of extractant, respectively. Under the optimized experimental conditions, the detection limit of the proposed method followed by ICP-OES was found to be 0.9?µg L^?1. The method was applied to the determination of copper in water and biological samples.     

Extraction,development and validation of HPLC-UV method for rapid and sensitive determination of colchicine from Colchicum autumnale L. Bulbs

Colchicum autumnale L. also known as the autumn crocus, contains colchicine alkaloid having antifungal properties. The tuber of this plant is rich in terms of colchicine. In this research an ultrasound-assisted extraction (UAE) method was optimized for the extraction of colchicine from Colchicum autumnale L. bulbs before high-performance liquid chromatography with UV detection (HPLC-UV). Optimization of various extraction parameters was performed using response surface methodology (RSM) to evaluate the maximum colchicine yield from Colchicum autumnale L. bulbs. The Box-Behnken design (BBD) and RSM were used to investigate the effect of three key parameters (extraction time (20–60?min), extraction temperature (40–80?°C) and ultrasound power (500–700?W) on extraction efficiency. The variance analysis suggested that the dependent response variable of yield of colchicine may be expressed by a quadratic polynomial model. The optimal theoretical extraction conditions were found to be an ultrasonication power of 602.4?W, an extraction time of 42?min and a temperature of 64?°C. Under these conditions, the optimum foreseen yield was 0.237%. The experimental colchicine yield obtained by following the optimized conditions was found to be 0.238%. These values are very well compatible with each other.