Production and molecular characterization of somatic hybrids between Pleurotus florida and Lentinula edodes.

Nine inter-generic somatic hybrids named as pfle were produced through PEG-mediated protoplast fusion between Pleurotus florida and Lentinula edodes using double selection method. Hybridity of the newly developed strains was established on the basis of colony morphology, mycelial growth, hyphal traits, fruit-body productivity and inter single sequence repeat (ISSR) marker profiling. Hybrid population was assessed with different phenotypic variables by one-way analysis of variance. Principal component matrices were analyzed for the six phenotypic variables in scatter plot showing maximum positive correlation between each variable for all strains examined. Six ISSR primers generated 66 reproducible fragments with 98.48 % polymorphism. The dendrogram thus created based on unweighted pair-group method with mathematic averages method of clustering and Euclidean distance which exhibited three major groups between the parents and pfle hybrids. Though P. florida parent remained in one group but it showed different degrees of genetic distance with all the hybrid lines belonging to the other two groups while L. edodes was most distantly related to all the hybrid lines. L. edodes specific sequence-rich ISSR amplicon was recorded in all the hybrid lines and in L. edodes but not in P. florida. All the fruit body generating pfle hybrid lines could produce basidiocarp on paddy straw in sub-tropical climate and showed phenotypic resemblance to the P. florida parent.

Molecular characterizations of somatic hybrids developed between Pleurotus florida and Lentinus squarrosulus through inter-simple sequence repeat markers and sequencing of ribosomal RNA-ITS gene.

The 12 pfls somatic hybrids and 2 parents of Pleurotus florida and Lentinus squarrosulus were characterized by ISSR and sequencing of rRNA-ITS genes. Five ISSR primers were used and amplified a total of 54 reproducible fragments with 98.14% polymorphism among all the pfls hybrid populations and parental strains. UPGMA-based cluster exhibited a dendrogram with three major groups between the parents and pfls hybrids. Parent P. florida and L. squarrosulus showed different degrees of genetic distance with all the hybrid lines and they showed closeness to hybrid pfls 1m and pfls 1h, respectively. ITS1(F) and ITS4(R) amplified the rRNA-ITS gene with 611-867 bp sequence length. The nucleotide polymorphisms were found in the ITS1, ITS2 and 5.8S rRNA region with different number of bases. Based on rRNA-ITS sequence, UPGMA cluster exhibited three distinct groups between L. squarrosulus and pfls 1p, pfls 1m and pfls 1s, and pfls 1e and P. florida.

A partially methylated mannogalactan from hybrid mushroom pfle 1p: purification, structural characterization, and study of immunoactivation.

A new water-soluble heteropolysaccharide (PS-II) with apparent molecular weight ∼1.65×10(5) Da, was isolated from the fruiting bodies of hybrid mushroom pfle 1p by hot aqueous extraction. It is composed of d-mannose, d-galactose, and 3-O-Me-d-galactose in a molar ratio of 1.0:0.99:1.1. The structural investigation of PS-II has been carried out using acid hydrolysis, methylation analysis, periodate oxidation study, and 1D/2D NMR experiments. Based on the results of these experiments, it was established that PS-II contained a main chain of (1→6) linked α-d-galactopyranosyl residues, one of which was substituted at C-2 by a terminal mannopyranosyl residue and also methylated at C-3 position. This heteropolysaccharide (PS-II) exhibited macrophage activation by NO production as well as in vitro splenocyte and thymocyte stimulation.

Glucan of a somatic hybrid mushroom, pfls1h: structural characterization and study of immunological activities.

A water-soluble glucan (PS-I) was isolated from the aqueous extract of the fruit bodies of a hybrid mushroom, pfls1h of Pleurotus florida and Lentinus squarrosulus (Mont.) Singer. Structural characterization of PS-I was carried out using total hydrolysis, methylation analysis, periodate oxidation, and NMR experiments ((1)H, (13)C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HSQC, and HMBC). Methylation analysis revealed that PS-I was composed of (1→3, 6), (1→3), (1→6)-linked and terminal ß-d-glucopyranosyl residues in a relative proportion of approximately 1:1:1:1. The repeating unit of the glucan consists of a backbone chain of two (1→6)-ß-d-glucopyranosyl residues, one of which is branched at O-3 position with (1→3)-ß-d-glucopyranosyl and terminated with a ß-d-glucopyranosyl residue. Study of immunological activity revealed that PS-I stimulates the splenocytes, thymocytes and macrophages.

Structural characterization of an immunoenhancing heteroglycan of a hybrid mushroom (pfls1h) of Pleurotus florida and Lentinus squarrosulus (Mont.) Singer.

An immunostimulating water-soluble heteroglycan (PS-II) was isolated from an aqueous extract of the fruit bodies of a hybrid mushroom, pfls1h produced by intergeneric protoplast fusion between Pleurotus florida and Lentinus squarrosulus (Mont.) Singer. Structural characterization of PS-II was carried out using sugar analysis, methylation experiment, periodate oxidation and 1D/2D NMR studies. Sugar analysis indicated the presence of glucose, mannose and galactose in a molar ratio of 1:1:2. Methylation analysis revealed that PS-II was composed of (1→6)- and (1→2,4,6)-α-D-galactopyranosyl, terminal ß-D-mannopyranosyl and terminal ß-D-glucopyranosyl residues in a relative proportion of approximately 1:1:1:1. On the basis of chemical analysis and NMR studies, the structure of the repeating unit of the heteroglycan was established to consist of a backbone chain of two (1→6)-α-D-galactopyranosyl residues, one of which is branched at O-2 with terminal ß-D-Manp and at O-4 with terminal ß-D-Glcp. This heteroglycan (PS-II) showed in vitro splenocyte, thymocyte and macrophage activations.

A heteropolysaccharide from an edible hybrid mushroom pfle 1p: structural and immunostimulating studies.

A water-soluble heteropolysaccharide (PS-I) having molecular weight ~2.1×10(5) Da was isolated from hot aqueous extract of the fruit bodies of hybrid mushroom pfle 1p. The hybrid mushroom pfle 1p was obtained through intergenic protoplast fusion between Pleurotus florida and Lentinula edodes. The heteropolysaccharide contained D-glucose, D-galactose, and D-mannose in a molar ratio of nearly 4:2:1. The structural investigation of PS-I has been carried out using sugar and methylation analyses as well as 1D/2D NMR experiments ((1)H, (13)C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HSQC, and HMBC). Based on the results of these experiments, the repeating unit of the PS-I was established as: [structure: see text]. PS-I showed in vitro macrophage activation by NO production and also stimulated splenocytes and thymocytes.

Structural characterization and study of immunoenhancing properties of a glucan isolated from a hybrid mushroom of Pleurotus florida and Lentinula edodes.

A water soluble glucan isolated from hot aqueous extract of fruit bodies of an edible hybrid mushroom Pfle1r of Pleurotus florida and Lentinula edodes showed macrophages, splenocytes, and thymocytes activation. The glucan consists of terminal, (1→3,6)-linked, and (1→6)-linked ß-D-glucopyranosyl moieties in a molar ratio of nearly 1:1:3. On the basis of acid hydrolysis, methylation, periodate oxidation study, and NMR studies ((1)H, (13)C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HSQC, and HMBC), the structure of the repeating unit of the glucan was established as: [structure: see text].