| Genetic diversity of 16 Phellinus strains based on rDNA ITS and RAPD markers, a series of research of Phellinus vaninii collected from Wanda Mountain such as biological characteristics, the molecular biological verification of taxonomic status, culture mode optimization of mycelia and fruitbody, nutritional components of fruitbody, mycelial polysaccharide, and inhibitory activity of the extracts to tumor cell proliferation, were studied by means of fungal science, ecology, molecular biology and biochemistry.(1) rDNA ITS sequences of 16 Phellinus strains were sequenced. The ITS1-5.8S-ITS2 lengths of 16 Phellinus strains are between 590 bp and 714 bp. The lengths of 5.8S are all 160 bp which is most conservative. The lengths of ITS 1 are between 220 bp and 310 bp, and the lengths of ITS2 are between 210 bp and 245 bp.10 Phellinus vaninii strains show an abundant genetic polymorphism by mean of RAPD markers, but the cluster analysis result of RAPD showed low relationship with geographical distribution.(2) Phellinus strain collected from Wanda Mountain was verified as Phellinus vaninii based on morphological identification and ITS sequence. The length of rDNA ITS sequence is 756 bp, GenBank number is HQ845057. Phellinus vaninii belongs to Basidiomycetes, Aphyllophomles, Hymenochaetacae, Phellinus. The geographical distribution area of the fungus is located in low altitude mountains of cold temperate zone (northern latitude 45°23’to 46°36’, east longitude 132° 11’to 133° 56’). The forest canopy closure is more than 55%, and the average tree-age of natural Populus davidiana and birch mixed forest is more than 50 years. The fruit body usually occur on sunny gentle slope or semi-sunny gentle slope where the forest canopy closure is higher and there are natural forest or wet meadow near the water. Its host is Populus davidiana.(3) The best growth nutrition for Phellinus vaninii collected from Wanda Mountain is as follows:carbon source is glucose, nitrogen source is wheat bran, inorganic salt is MgSO4. The effect order of nutritional factors on mycelial growth (dry weight) is:wheat bran>glucose> MgSO4 (solid culture), glucose> MgSO4>wheat bran (liquid culture). The better interaction effect groups for mycelium growth are glucose+wheat bran and glucose+MgSO4. Under the optimum formula of solid culture (potato 200 g/L, agar 20 g/L, glucose 30 g/L, wheat bran 15 g/L, MgSO42 g/L, KH2PO43 g/L; pH 6.5,28℃, dark, liquid volume 280 mL in 500 mL Erlenmeyer flask), the maximum dry weight of mycelia is 12.9 g/L. Under the optimum formula of liquid culture (potato 200 g/L, glucose 30 g/L, wheat bran lOg/L, MgSO45g/L, KH2PO43g/L, VBi 100 mg/L; pH 6.5,28℃,170 r/min), the maximum dry weight of mycelia is 14.5g/L after 12d culture.(4) The best fruiting mode of substitute material culture for Phellinus vaninii collected from Wanda Mountain is the "both ends fruiting from the bag wall", "the diamond-shaped hole on bags". The optimum formula for substitute material culture is:73% oak wood chips,5% poplar wood chips,8% wheat bran,8% rice bran,1% gypsum,2% corn starch, and 1% calclime.The water content of the culture medium is 60%-65%. The pH value of the culture medium is 6.5. Based on this culture mode, the optimum conditions for fruiting are:the height and width of plastic fruiting shed are not more than 2 meters and 5 meters respectively, loam soil ground, there are ditches interlinked outside, scattered light in shed is 300 lx. In bud-stage the temperature is 21℃～23℃, relative humidity is 80%-85%, and in fruiting stage the temperature is 22℃～25℃, the relative humidity should be 90%. To wood-log culture, the height and weight of plastic fruiting shed are not more than 1.7 meters and 5 meters. During the bud-stage, the temperature should be 21℃～23℃, relative humidity is 80%-85%. But for fruiting the temperature shoud be 22℃～25℃, and the relative humidity should be 90%-95%.(5) Three kinds of polysaccharide were isolated from mycelia of Phellinus vaninii collected from Wanda Mountain. Major components of polysaccharide S1 and S3 include glucose, galactose and mannose. S1 and S3 are all glucan, but their structures are different. S1 is furanose form and S3 is pyranose form.(6) The submerge fermentation of Phellinus vaninii collected from Wanda Mountain was done. The rheological properties of fermented liquid, such as viscosity, pH, residual sugar content and polysaccharide production, were studied. The results show that the parameters in the fermentation process have the same variation trend with mycelia dry weight. The parameters in the fermentation process all reach the peak after 8 days fermentation. We also studied the inhibiting effects of different extracts, which extracted from fermented liquid, on tumor cell proliferation. The rusults show that the extracts have different inhibiting activities to tumor cell proliferation. Ethyl acetate extract has the best inhibiting avtivity. The inhibitory rate of the extract (1000μg/mL) is 87.5% after 48 h. IC50 of the extract is 53.459μg/mL. The inhibitory rates of IC50 are 90% and 95%, respectively, after 96 h, and 120 h. |
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