Separation, Purification, Composition Analysis And Inactivation Of The Toxin Of Rhizoctonia Solani

Different methods were used to extract the crude toxin from the culture filtrate of Rhizoctonia solani causing rice sheath blight, in which the most appropriate method as follows: when the culture filtrate was concentrated to one fifth of the original volume at 60℃, the equal volume of methanol was added, and then the precipitate was removed by centrifugation and methanol was evaporated with rotavapor at 60℃. The water fraction was extracted with the equal volume of ether for three times. The ether fraction was mixed and evaporated with rotavapor at 40℃, and the crude toxin was obtained. The biological activity assay of the crude toxin showed that it could obviously inhibit the growth of rice radicle and induce the characteristic symptom of rice sheath blight.The crude toxin extracted by different methods showed similar UV absorption spectra, and the maximum absorptive wave length was about 220nm. The crude toxin was seprated into 43 fractions by first Sephadex G-75 silica gel column chromatography, in which the eluate of 7th~18th bottles was bioactive. After the eluate first obtained was purified in the same way on the second time, the eluates of 21 bottles were collected, and that in 7th~9th bottles had the strongest activity, which was called the pure toxin.By HPLC analysis, the pure toxin had at least four specific absorption peaks, which indicated that the toxin could contain 4 active components. Through IR spectrum determination, the pure toxin could contain N-H (or O-H), C = O, CN (or C-O) signals. On the basis of TLC analysis, the toxin was carbohydrate, which contained glucose, N-acetyl-mannose and sucrose according to GC-MS spectrometry. But, benzoic acid, phenylacetic acid and their ramifications were not found in the toxin. When 9 kinds of compounds were mixed with the crude toxin at a certain proportion (100, 500, 1000μg/ml), some compounds could significantly inactivate the toxin according to rice radicle growth inhibition assay,in which KMnO4 had the most inactivation with the inactivation rate of about 80%. When the crude toxin inactivated by KMnO4 was used to inoculate rice sheath, the symptom was significantly reduced in comparion with the toxin not treated, that is, only brown spots appeared and necrotic spots were not found.There was a significantly positive correlation between the ability of toxin production and the virulence of R. solani. which indicated that the toxin of was an important pathogenic factor.