Screening Of Fungi Strains For Biological Control On Meloidogyn Incognita And Assay Of Their Metabolites

Fungi strains for biological control on Meloidogyn incognita were isolated from soil samples collected from Qinghai province using M.incognita as target.The fungal strains which were selected were identified according to traditional classification and molecular phylogenetic.The biological active metabolites produced from fungi strains were purified and assayed using silica gel column chromatography,silica gel thin-layer chromatography and other chromatography methods.At the same,the chemical structures of the active metabolites were assayed by MS and NMR analysis.The LC50 values of the active compounds produced by two fungi strains were determined by laboratory toxicity methods and the effects of the strains for control cucumber Meloidogyn incognita were investigated by pot experiments.The results were as follows.1.By preliminary screening and secondary screening,two fungi strains,named as MHZ111 and D35,respectively,showed high and stable antinematicidal activity against Meloidogyne incogonita,the lethal rate was more than 90.0%.They were identified as Penicillium purpurogenum and Penicillium aff.sclerotiorum by analysis of morphological characteristic and ITS sequencing.2.The active metabolites which were produced by strains MHZ111 and D35 were isolated and purified as follows.?1?Extraction: The metabolites were extracted using ethyl acetate,then the extract was concentrated by the rotary evaporator.?2?Silica gel column chromatography: The concentrated sections were mixed with 200³00 mesh silica at the mass ratio of 1:5,the mixture was dried and porphyrized and mixed and packed column in order,the volume of eluent is four times of the volume of silica gel column,the types of eluant were petroleum ether,petroleum ether/acetone?20:1,10:1,5:1,1:1;v/v?,acetone,methylene chloride/methanol?20:1,10:1,5:1,1:1;v/v?and methanol.Gradient elution chromatography was applied to separate and purify the active metabolites under vacuum condition,the eluant was collected once per 250 m L and concentrated by the rotary evaporator.?3?TLC: Each grope concentrate was used to do the TLC experiment using petroleum ether/acetone?1:1;v/v?as developing solvent,the nematicidal active ingredient was combined,which had the same Rf value.?4?Cyclic preparation: The compositions which had nematicidal activity and the same Rf values were prepared by recycling preparative HPLC.Finally,the single active compound was obtained.3.Six active compounds were obtained from the metabolites which produced fromstrains MHZ111 and D35.Their structures were analyzed by MS and NMR.The results showed that the chemical formula for the four compounds produced by strain MHZ111 were C₂₅H₂₈O₇,C₂₆H₃₂O₈,C₂₅H₃₀O₆,and C₁₈H₁₆O₆,respectively,and named as Miscellaneous terpenoids,22-epoxyberkeleydione,Chrodrimanin E and Vermistatin;the chemical formula for the two compounds produced by strain D35 were C12H12O4 and C20H24O9,and identified as 2-methyl-5,7-methoxytryptamine ketone and Double-heptyl phthalate ester,respectively.4.The laboratory toxicities of six biological active compounds were determined,the results showed that,the six compounds showed strong nematicidal activity.The LC50 values of four compounds obtained from the metabolites produced by strain MHZ111 which were Miscellaneous terpenoids,22-epoxyberkeleydione,Chrodrimanin E and Vermistatin were109.5 mg/L,95.4 mg/L,117.1 mg/L and 88.7 mg/L,respectively.The LC50 values of two compounds produced by strain D35,which were 2-methyl-5,7-methoxytryptamine ketone and Double-heptyl phthalate ester,were 179.2 mg/L and 177.2 mg/L,respectively.5.The pot experiments were conducted by using the fermentation filtrate of strains MHZ111 and D35 treating cucumber seedlings.Results indicated that the root-knot nematode was significantly lighter than that of control group.When the cucumber seedlings were treated by the fermentation filtrate of strain MHZ111,the disease index of cucumber Meloidogyne incognita was 25.4,control efficacy was 73.8%.When the cucumber seedlings were treated by the fermentation filtrate of strain D35,the disease index of cucumber Meloidogyne incognita was 22.2,control efficacy was 77.1%.