Abstract:It was found that Pestalotiopsis microspora PM-1, which was isolated from the infected moso bamboo, had higher herbicidal activity. In order to make certain the optimal liquid fermentation conditions for producing the herbicidal substance, different media, culture temperatures, culture time and culture mode were researched and the herbicidal substance was separated using TLC and HPLC . The bioactivity reached the highest when PM-1 isolate was cultured statically in PD medium containing ferrous sulfate at 30 ℃ for 15 days in the darkness. TLC analysis and the bioassay results indicated that band 6 had the strongest activity among the 7 bands. Seven fractions were obtained by analysing to band 6 through HPLC and fraction 3 was found to be a pure compound with high activity to Digitaria sanguinalis with the retention time at 46.3 min. These results approved the conclusion that a herbicidal compound, fraction 3, was obtained when PM-1 was cultured under the optimal liquid fermentation conditions.