Studies On The Mutation Breeding And Antifungal Mechanism Of Penicillum Sp TS67

The marine fungi TS67(Penicillum sp.) isolated from Bohai Sea has broad spectrum resistance to plant pathogenic fungi.Aimed at Bipolaris maydis and Fusarium oxysporum,the mutated breeding of high-yielding mutants,physicochemical property and type of active substance,the antifungal mechanism of the TS67(Penicillum sp.)were studied in this paper.(1) In order to improve the antifungal activity,the spores of Penicillum sp TS67 as a original strain were mutagenized by physical and chemical mutation.After using method of the dual culture and Oxford plate,three high-yielding mutants Z10,W8,UD-3 were obtained,the result of antifungal activity test indicated that their activity was increased by 41.9%,45.6%,31.3%compared with the original strain.Through fermentation experiment in shaking flasks,heredity character of three high-yielding mutants was stable.(2) The antifungal mechanism of the TS67(Penicillum sp.) was investigated,and the test indicated that mycelia growth,spores forming and spores germination of Bipolaris maydis and Fusarium oxysporum can be inhibited by metabolites produced by TS67, after 50%fermentation broth of strain TS67 acting 120 hours,the inhibition rate of hyphal growth can reach about 77.78%,70.30%,the inhibition rate of spores forming also arrived at 58.8%,73.5%;under 50%fermentation broth acting 12 hours,the inhibition rate of spores germination were up to 78.3%,62.0%,the mycelia grown under active metabolites also showed surface nodulation and growing point expanding irregularly, protoplasmic condensing etc.These results showed that activity metabolites of strain TS67 maybe target at fungal cell wall.The test of physicochemical property and purification indicated that antifungal substance is protein.(3) The enzyme activity of chitinase andβ-glucanase produced by Penicillum sp.TS67 were determined by 3,5-dinitrosalicylic acid method,the enzyme activity of them were 2.2 U/mg and 13.1 U/mg.The optimal temperature and pH ofβ-glucan enzyme are 55℃and 5.0 respectively.Theβ-glucanase was more stable at low pH than at high pH,and was relatively stable below 60℃.Some metal ion K⁺,Mg²⁺,Ca²⁺,Al³⁺, Mn²⁺,Fe²⁺,Fe³⁺,Zn²⁺could stimulate the enzyme activity,β-glucanase molecular mass was estimated to be about 62 kD by SDS-PAGE.