Initial Separation Of Herbicidal Substances Of Metabolites Produced By Pythium Aphanidermatum

Microbial herbicides, arouses more and more attention with their environmental compatibility and the advantages of high security. Pythium aphanidermatum is a kind of pathogen belongs to Oomycete, which can cause many diseases such as bed rot of many kinds of crops, melons and fruits. The previous results showed that the crude toxin of P. aphanidermatum had strong herbicidal activity. In this study, herbicidal active substance was separated and purified from P. aphanidermatum PAC which was isolated from the cucumber. P. aphanidermatum was cultured in PD medium and the culture filtrate was extracted with ethyl acetate, then the ethyl acetate extracts were separated gradually by silica gel column. The mycelia were extracted with ethyl acetate and methanol, and the methanol extracts were separated gradually by silica gel column.The ethyl acetate extracts were separated by silica gel column using different proportion of ethyl acetate and petroleum ether(V/V=4:1,3:1,2:1,1:1,1:2,1:3,1:4). Eluents were collected and each 50 mL were considered as a fraction and bioassayed using the method of growth inhibition and seed germination. The results showed that when the crude toxin was well chromatographyed with the developer of ethyl acetateand petroleum ether(V/V=3: 1) and fraction 8 had stronger inhibitory activity against Digtaria sanguinealis, which reached level 3 and the seed germination inhibition rate was 13.64%. The mycelia extracts of ethyl acetate were separated by silica gel column using different proportion of ethyl acetate and petroleum ether(V/V=4:1,3:1,2:1,1:1,1:2,1:3,1:4). Eluents were collected and each 50 mL were considered as a fraction and bioassayed using the method of growth inhibition and seed germination. The results showed that the crude toxin was well separated with the developer of ethyl acetateand petroleum ether (V/V=4: 1) and fraction 11 had the stronger inhibitory activity against Digtaria sanguinealis, which reached level 3 and the seed germination inhibition rate was 17.39%.Methanol extracts of mycelia were separated by silica gel column using different developers (ethyl acetate:petroleum etherV/V=4:1,3:1,2:1,1:1,1:2,1:3,1:4). Eluents were collected and each 50 mL were considered as a fraction and bioassayed using the method of growth inhibition and seed germination. The results showed that the crude toxin was separated well with the developer of and fraction 4 had the stronger inhibitory activity against Digtaria sanguinealis, which reached level 5 and the seed germination inhibition rate was 68.97%. The results revealed that fractions 21~24 obtained by gradually eluting with ethyl acetate and petroleum ether (V/V=3:1 and 2:1) showed strong activity against Digtaria sanguinealis with the inhibition level of 4. The combined fractions of 21~24 were eluted with the mixture of petroleum ether and ethyl acetate (V/V=2:1) as the developer and 20 fractions were collected. Bioassay results showed that fraction 3 had the stronger inhibitory activity against Digtaria sanguinealis.HPLC analysis suggested that this fraction mainly contained 3 constituents, the retention time of which was 12.7 min, 14.0 min and 30.5 min respectively. The results of Mass Spectrometry showed that the likely formula of constituent 2 and 3 was C24H35N3O3,C20H23N9O3 and C22H25N6O4,C19H37N6O2å’ŒC22H33N6O2,respectively.