Research On The Inhibiting Substances Of Hypoxylon Perforatum And Their Effects On Sphaeropsis Sapinea

The effects of inhibiting active substance extracted from culture liquid of Hypoxylon perforatum on Sphaeropsis sapinea;isolation,purification and structure identification of inhibiting active substance; ecological safety test and stability of inhibiting active substance;the rDNA ITS sequences of Hypoxylon perforatum and Sphaeropsis sapinea were studied by methods of Phytopathology, Molecular Biology,Biochemical and Organic Chemical.The results showed as follows:(1) rDNA ITS sequences were amplified from Hypoxylon perforatum and Sphaeropsis sapinea strain with the universal primers ITS1 and ITS4.rDNA ITS sequence lenth of Hypoxylon perforatum is 617bp and GenBank Accession is EU744544;rDNA ITS sequence lenth of Sphaeropsis sapinea is 544 bp and GenBank Accession is EU796887.Through the BLAST In GenBank,the strains were confirmed Hypoxylon perforatum and Sphaeropsis sapinea.(2) Methods of water-extract and ester-extract were used for extracting active substances from culture liquid of Hypoxylon perforatum.Method of water-extract included two ways,one is extracting directly from cultural liquid,and the others is extracting by ultrasonic.N-butyl alcohol, ethyl acetate and alcohol were used as extractant in ester-extract test.Growth-inhibiting rate and germination-inhibiting rate were used as the index of antifungal activity.Growth rate method was used for determining growth-inhibiting rate and pendent drop method for determining germination-inhibiting rate.The results is that five extracts from culture liquid of Hypoxylon perforatum all have definite antifungal activities to Sphaeropsis sapinea growth and germination.Ethyl acetate-extract is the best one on inhibiting Sphaeropsis sapinea growth,the growth-inhibiting rate is 73.20%;the germination-inhibiting rates of ethyl acetate-extract,ultrasonic-extract and n-butyl alcohol-extract are all over 90%.(3) The result of isolation,purification of inhibiting active substance shows that there are three components in ethyl acetate-extract.Component I has the highest inhibiting effect on Sphaeropsis sapinea growth,the growth-inhibiting rate is 72.94%.Component I has three compounds,one is Methoxy-cinnamic acid methyl ester whose yield is highest(57.952%),the other two are iso-butyl phthalate(yield is 13.592%) and dibutyl phthalate(yield is 28.455%).(4) Ultraviolet rays,temperature,pH,oxidants and reductant,storage time were selected as actors of the stability of ethyl acetate-extract.In the natural environment,ethyl acetate-extract has a high stability to ultraviolet rays and temperature,definite anti-oxidation and anti-reduction capacity, and good heat resistance.Long-term storage does not affect its antifungal activity.(5) In the oral acute toxic examination,the mice'death did not happen.The LD₅₀ of the extraction is less than13200mg/kg,which was micro-toxic.