| The effects of toxic mushroom strains and inhibiting active substance extracted from culture liquid of A. virosa on C. chrysosperma were studied by methods of Phytopathology, Molecular Biology, Biochemical and Organic Chemical. We also studied the structure and characteristic of the inhibiting activity, the control mechanism of A. virosa. The results showed as follows:(1) A. virosa screened from 9 toxic mushroom strains would been farther studed as excellent effect strains on C. chrysosperma. The activity substances exist in culture liquid or mycelia, the effect of liquid inhibiting by shakering is higher than that by stationary culture, extracts by n-butyl alcohol and ethyl acetate have mighty significant difference(a=0.01) with CK; water and alcohol are the best solvents for extracing inhibiting activity substance from mycelia; Ultrasonic extracting form is the best one to both mycelia and culture liquid. Inhibiting activity of extracts descent when treated by autoclaving.(2) By methods of Sephadex isolation, FT-IR spectrometry, UV spectrumscopy and LC-MS, the research on structure of inhibiting active substances of A. virosa were done. The results show that the inhibiting active substance have 5 compositions which are amide or mixtures of amide, and all of which have high inhibiting effect on C. chrysosperma; composition A have 2 kind of amides which the molecular weight respective was Al 5732.39 and A2 7187.01; composition B is a kind of amide which the molecular weight is 5186.57, composition C have 3 kind of amides which the molecular weight respective is C1 5336.44, C2 1057.99 and C3 1877.57; composition D have 3 kind of amides which the molecular weight respective is D1 3118.51, D2 3046.40 and D3 1877.57; composition E had 3 kind of amides which the molecular weight respective is E1 1185.30, E2 3099.95, E3 3558.47. The basal structure cell of B and C1 were determined.(3) The control effect and secuiruty assessment of inhibiting active substance of A. virosa on C. chrysosperma were studed, the results show that the crude extractive and composition IV have evdent prevention effect on pathogen and the prevention effect are 87.0%; in disease prevalent period, the crude extractive and composition IV had diffinite prevention effect and which respective is 34.8% and 41.3%. LD50 of the mycelium of C. chrysosperma is 418.70 mg/kg (95% credible limit was 453.11-386.90 mg/kg) and is low-toxic; the crude extractive and composition IV is ecological security.(4) Stability and ferment condition of inhibing activity substance of A. virosa were studied. Carbon source is the key factor of affacting the biomass and inhibiting active of inhibing activity substance. To mycelium growth and inhibiting activity substances producting, the optimum carbon source is lactose; the optimum nitrogen sourse is peptone; the optimumculture medium recipe is: lactose 3％, peptone 0.5％, MgSO4: 0.15％, KH2PO4: 0.45％. Lactoseis the most important factor for inhibiting activity substances producting. The optimumferment condition are pH 5～6, temperature 25℃～30℃, inoculation capacity about 1％, propercapacity of culture liquid 50％, optimum shaker speed 120 r/min～160r/min and culturedtime15d～20d. The critical temperature is＞75℃, the critical pH is＞9 or＜4, and inhibitingactivity substance would been negative influenced when the time of UV irradiation is exceed4h.(5) Inhibiting machanism of A. virosa on C. chrysosperma were researched on somekey enzyme energy varing. The inhibiting active substance of A. virosa can make the energy ofHexokinase descent, the energy of Lactate Dehydrogenase, SDH dehydrogenase, PyruvateKinase and Adenosine Triphosphatase increase, then promote the respiration and produce moreReactive Oxygen Free Radicals, and pathogen's biofilm are demaged. The contents of 3 kindsof enzyme of biophylacxis are very low, and clearing ability itself is low also. Many ReactiveOxygen Free Radicals injury biomolecules for they can not be cleared in time. The injurydegree increase with the concentration of A. virosa crude extractive.(6) Classification and physiology characteristics of A. virosa and C. chrysosperma werestudied, rDNA ITS sequence lenth of A. virosa is 516bp and GenBank Accession is EF403032;the optimum culture temperature is 30℃, the optimum pH is 5～7, the optimum carbon sourceand nitrogen source respective is Mannitol and Yeast; the optimum culture medium recipe is:patato 200g, glucose 20g, MgSO4 1.5g, NaH2PO4 3g, agar 20g and water 1000mL. rDNA ITSsequence lenth of C. chrysosperma is 907bp and GenBank Accession is EF101159; theoptimum culture temperature is 25℃, the optimum pH is 5～6, the optimum carbon source andnitrogen source respective is glucose and peptone; the optimum culture medium recipe is:patato 200g, sucrose 20g, yeast 2g, peptone 2g MgSO4 0.5g, KH2PO4 3g, agar 20g, VB1100mg and water 1000mL.Under indoor artificial culture condition, the spore sprouting of C. chrysosperma havephenological period, the ragularity of spore producing is as the same as that in nature, and thegrowth of mycelia had no change with seasons turning. |
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