Studies On Sclerotial Formation By Pure Culture And Mechanisms Of Sclerotial Transformation Of Polyporus Umbellatus

Polyporus umbellatus（Pers.） Pil（?）t,as a sclerotia-forming mushroom,belongs to the family Polyporaceae in the class Basidiomycetes.Sclerotia of P.umbellatus is one of the commonly used and precious medicinal fungi,with sclerotia being widely used for many years as an antidiuretic agent as well as an antidote in oriental countries,especially China, Korea and Japan.Since the 1970s,when one kind of water-soluble polysaccharide from the sclerotia was found to enhance immune activity in humans and have a good anti-tumor effect,investigations on chemical and nutritional constituents of P.umbellatus have been carried out in many countries.It was also reported that sclerotia of P.umbellatus had potent effects,suppressing the cytotoxic activity of leukemia and hepatitis.This investigation covered such a few aspects of P.umbellatus:the nutritional conditions influencing mycelial growth and sclerotial formation,activities of key enzymes in the carbon catabolism pathway,the relationship between reactive oxygen species（ROS） with sclerotial formation,the signal transduction way during the morphological transformation of P.umbellatus,and the conditions influencing mycelial protoplast production and regeneration of P.umbellatus.The nutritional requirements of 15 carbohydrates,10 nitrogen compounds,8 vitamins,and 8 mineral elements were studied for their effects on mycelial growth and sclerotial formation in agar cultures of P.umbellatus using the one-factor-at-a-time method.Among the nutritional factors tested,only fructose as the sole carbon source could induce sclerotial formation of P.umbellatus.An additional test indicated that nitrogen source categories influenced sclerotial formation significantly and sclerotium only formed in treatments with complex organic nitrogen.Peptone was found to be the best for sclerotial production.Through an orthogonal matrix test,the effects of carbon/nitrogen factors on sclerotial formation were found be in the order:fructose＞interaction between fructose and peptone＞peptone.The optimal concentration for sclerotial formation was determined to be 50.0 g/L fructose and 4.0 g/L peptone.The concentrations of mineral salts including K₂HPO₄、KH₂PO₄ and MgSO₄ were optimized for the sclerotial formation as 1.0g/L,0.5g/L and 0.5g/L.In the further studies on the interaction between fructose and peptone,it was found that the mixture of fructose and peptone being moist heat sterilized together could reduce the time from mycelial growth to sclerotial initiation and enhance the sclerotial production of P.umbellatus compared with the fructose and peptone being moist heat sterilized separately.The mixture of fructose and arginine being moist sterilized indicated the same effect on P.umbellatus.But the effects of different concentrations of arginine was just opposite,the high concentration in this study of 3.0g/L inhibited the sclerotial formation completely,the low concentration of 1.0g/L promoted the sclerotial yield greatly compared the optimized fructose complete medium.Morphological characteristics of the sclerotium forming on the fructose complete medium were investigated.The light microscopy indicated the artificial sclerotium was made of the hyphae in a highly differentiated and organized state.In addition,the artificial sclerotium had the same fingerprint compounds:polysterone A and polysterone B of the wild sclerotium according HPLC analysis.The difference of utilization on carbon sources:fructose and glucose by P.umbellatus was investigated by analysis of key enzyme activities on the carbon catabolism pathways.Glycolysis of fructose and glucose in P.umbellatus was same as the cooperation and HMP and EMP pathways.The activity of HMP key enzyme, 6-phosphate gluconic acid dehydrogenase,was significantly higher than the EMP key enzyme,phosphate fructose kinase.This leaded to the conclusion:HMP was the main way of glycolysis of P.umbellatus.But the two enzyme activities of mycelia with fructose as the carbon source was apparently greater than mycelia growing in the glucose complete medium.In addition,the enzyme tests indicted the action of glycoxylate cycle was not so important as the report of other sclerotium-forming fungi and tricarboxylic acid cycle was the main pathway of P.umbellatus for carbon aerobic digestion.Aerobic respiration degree of mycelia with fructose as the carbon source was also greater than mycelia growing in the glucose complete medium.According to the test of residual fructose in the medium during growth of P.umbellatus,the utilization of fructose became greatly fast before sclerotial formation and the fructose content almost lowered to none as the sclerotium developed completely.In this study,it was found 3 antioxidants-Vc,Ve and oxalic acid could influence the sclerotial formation greatly.Their effects were same with the mixture of fructose and peptone being moist heat sterilized,the high concentration in this study inhibited the sclerotial formation completely,the low concentration promoted the sclerotial yield greatly compared the optimized fructose complete medium.Superoxide in mycelial cell of P.umbellatus was detected and assayed by NBT color-developing method.The light microscopic images and detailed test data indicated the mycelial growth rate,carbon source category and Ca²⁺ signal inhibitors all could influence the mycelial superoxide content greatly.The interesting point was the mycelial superoxide reduced greatly after the sclerotial transformation.During moist heat fertilization,there took place the mailard reaction between fructose and arginine and the one important product- melanoidin is also a highly antioxidative compound.Melanoidin was extracted from the water solution of fructose and peptone being moist heat sterilized together.Melanoidin of different concentrations was added to the fructose complete medium with fructose and peptone being moist heat sterilized separately.Its effect on P.umbellatus was the same as Vc,Ve and oxalic acid.Growth and morphogenesis transformation of P.umbellatus were examined in the presence of various pharmacological compounds to investigate signal transduction pathways that influence the development of sclerotia.Calcium channel blocker nifedipine and calcium ionophore A23187 reduced sclerotial formation of P.umbellatus.Then,3 classes of Ca²⁺ signal agent including calcium chelator,calcium channel blocker and calcium ionophore were further studied for their effects on sclerotial formation with different concentrations.Among them,EGTA and BAPTA as calcium chelator exhibited complete inhibitory effect on sclerotial formation among the levels tested.Calcium channel blockers and calcium ionophore at the concentrations of this study neither could eliminate sclerotia formation entirely,but inhibited such transformation greatly. Notoginsenoside over 250μg/ml produced the significant negative effect on the mycelial growth and prevent sclerotial formation thoroughly at the dosage of 500μg/ml,whereas other drugs didn’t influence vegetative growth at all.As the calcium ionophore,A23187 at low dosage（20 nM） didn’t decrease sclerotial mean weight.Only at the high levels of 200 nM,A23187 showed negative effect on sclerotial formation,but not completely.The inhibitory effects of Ca²⁺ antagonists could be reversed partially or completely by the addition of Ca²⁺.Using Fluo-3/AM as the fluorescent indicator of cytosolic free calcium, the Ca²⁺ content in the cytoplasm decreased significantly when hyphae were treated with different drugs,and there is no active Ca²⁺ signal in sclerotial mycelium.Taken together, the results suggest that Ca²⁺ signal transduction may play an important role in sclerotial formation of P.umbellatus.The condition influencing mycelial protoplast production and regeneration of P.umbellatus was investigated.The best way for both the protoplast production and regeneration was:mycelia by liquid culture of P.umbellatus after 10d was gained and reacted with 15mg/ml lywallzyme（pH6.0,0.6M sucrose water solution） under 32℃for 2h.Then the protoplast could regenerate successfully in liquid malt medium(pH6.0, 0.6M sucrose water solution.