Preparative Separation Of The Herbicidal Substance From Pestalotiopsis Microspora PM-1Isolate

One strain was isolated from the infected leaves of bamboo and it was determined tobe Pestalotiopsis microspora PM-1by the molecular and morphological identification. Theprevious research showed that the crude extracts from Pestalotiopsis microspora PM-1hadhigher herbicidal activity on weeds and might be developed to be bioherbicide. Thisresearch is focus on separating and purification of the herbicidal fractions and the mainresults are as f ollows:1. PD media (Potato dextrose broth) was used to culture the Pestalotiopsis microsporaPM-1, and the filtrate liquid was extracted by ethyl acetate with the same volume andevaporated under reduced pressure to obtain the crude extracts. The high-speedcountercurrent chromatography (high speed counter current chromatography, HSCCC) wereused for separation and purification of the crude extracts. In order to select the best HSCCCsolvent system for crude extracts,10kinds of solvent systems were prepared according tothe method designed by Oka. The results showed that the method of seed germination wassuitable to determine the partition coefficient of the solvent system. The results alsoshowed that No.7solvent system was more suitable for HSCCC separation of crude extracts.The ethyl acetate crude extracts of PM-1were separated by HSCCC with the two-phasesolvent system consisting of n-hexane-ethyl acetate-methanol-water-solution (4:5:4:5) andthe results showed that the crude extracts can be preliminarily separated as Ⅰ、Ⅱ、Ⅲ andⅣ four componentsof whichfractionⅡ had the most intensive herbicidal activity onDigitaria sanguinalis and was further analyzed by high performance liquid chromatography(HPLC) on C18column with acetonitrile:water=75:25(V/V) as the mobile phase underisocratic elution. Fraction A with the retention time of7.954min showed an effectiveherbicidal activity on seed germination of Digitaria sanguinalis and theby DAD analysisindicated that fraction A is a pure compound.2. The crude extracts were separated by silica gel column chromatograph, and HPLC. The results showed that the crude extracts can be separated for two components. Fraction Bwith the retention time of3.510min has the most herbicidal activities, and it was proved tobe a pure compound by. the DAD analysis.)...