Extraction And Activity Determination Of Toxin Fromendothia Parasitia

In recent years, Chinese chestnut blights incidence density was rates and higher and to make that chestnut trees had more and more serious harm, caused substantial economic losses.The study of chestnut blight pathogenic toxins became increasingly important. In this study,Endothia parasitic virulent strain Cfcc82606 was based on research, explicated the basic physical and chemical properties of Endothia parasitic toxin, by adopted the different organic solvent extractions to obtain crude toxin,and used silica gel column chromatography to separation and purification of toxin from it many times, and Used gas phase mass spectrometry（GC-MS） for structure analysis of separation and purification of toxin identification. The main results of the study were as follows:1. The crude protein toxin and non-protein toxin were prepared by methanol precipitation from the filtrate of Endothia parasitica（Cfcc82606）. Analysised of the dialysis bag and biological activity detection, suggested that the toxin belongs to a s non-protein,small molecular substances. The changes of pathogenic activity of non-protein components after the treatments of the different temperature, different p H value and different ultraviolet irradiation time. The results showed that the toxin had a strong thermal stability,and still had a strong pathogenic activity after after treated with high pressure, high temperature for30min;The non-protein toxin was sensitive to p H values, and would be increased under acidic conditions, weakened under alkaline conditions; The ultraviolet radiation could reduce the toxicity of the toxin. After passed through 7 different polar organic solvent extraction,.Detected that the n-butanol in organic phase had the strongest pathogenicity, inferred Endothia parasitica toxin may be polar substances. Furthermore, Endothia parasitica Cfcc82606 strains of non-protein toxin had a various degress of pathogenicity on the seedings’ growth of different plant species, indicating that the non-protein toxin was a non-host specific toxin.2. Endothia parasitica（Cfcc82606） culture filtrate was extracted by taken different organic reagents, the results showed: the highest rates of toxin extraction method: after non-protein toxins to be processed has been adding 1.5 times three times the volume of n-butanol extracted,merged in organic phase,40℃ rotary evaporation to remove n-butanol,37℃ dried, got the Endothia parasitic crude toxin. Determination of the crude toxin for biological found chestnut leafy branches had the same symptoms as the toxic liquid, and the time and disease index was higher than that in crude toxin3. Crude toxin was extracted by four different polar organic solvent, Different toxin’s organic solvent extraction is scanning UV spectrophotometer, which has a similar UV absorption curve and the peak was around at 237 nm, illustrated that the toxin active ingredients was no influenced by organic solvent.Crude toxin eluent screened different solvents of different polarity proportion, determine the selection of chloroform: methanol:water（9: 2: 0.1,v/v）as a crude toxin silica gel column chromatography eluent The resulting crude toxin with chloroform: methanol: water（9:2:0.1,v/v） as eluent to silica gel column chromatography, collecting a total of 106 bottles of the eluent, after testing, the main active component 11¹9 bottles, bottles 31⁴2, 51⁵6 bottles, 68⁷6 bottles. The eluate containing the active ingredient several times after elution, to give 3 pure toxin, toxin named polarâ… ,polarâ…¡, polarâ…¢.4. Biological activity detected of three pure toxin, the three toxins were had pathogenic on chestnut leaves branches, but had different symptoms. Measured by GC-MS analysis showed that the polar â… was 2,5-Dioxotetrahydrofuran, molecular size was 100, the formula was C₄H₄O₃. Polar â…¡was Dibutyl phthalate, molecular size was 278, the formula was C₁₆H₂₂O₄; Polar â…¢ was 5-Hydroxymethylfurfural, molecular size was 126, the formula was C₆H₆O₃. Toxin chemical structural formula were:5-H ydroxym ethylfurfural Polarâ… Polarâ…¡ Polarâ…¢5. Different biological detection of three pure toxin were found,three kinds of toxins had pathogenic effect on the branches and leaves of chestnut and pathogenicity index was higher than that of crude toxin and culture filtrate toxin,but there are some differences in the conditions of the disease.It showed that the role of toxin in plant pathogenic, not onlya single material effects on the plants, but also metabolic product of the result of different pathogenic substances.