Identification of allelopathic substances secreted from the bacillariophycean Cylindrotheca closterium inhibiting the growth of the red-tide raphidophycean flagellate Heterosigma akashiwo

We previously proposed that growth inhibition of Heterosigma akashiwo (raphidophyceaen) during mixed cultured with Cylindrotheca closterium (bacillariophyceaen) might be brought about by an allelopathic substance produced by C. closterium. In this paper, we reconfirmed the allelopathic phenomenon to be caused by an algicidal substance secreted from C. closterium and then identified the allelopathic substances to be fatty acids such as cis-6,9,12,15- octadecatetraenoic acid (ODTA). The cell numbers of H. akashiwo in a unialgal-culture in medium filtrated with a glass fiber filter from a unialgal cultures of C. closterium under the initial stages of the stationary growth phase (non-treated medium) decreased significantly to approximately 5% of that in Erd-Schreiber modified medium (control) within 72 hours after the beginning of culture. The growth inhibition was unaffected when the culture mediums were autoclaved or nutritive salts were added to the non-treated medium, and when both treatments were combined. From these results, together with the failure to detect eicosapentaenoic acid in the non-treated medium, it was strongly suggested that the growth inhibition of H. akashiwo was caused by an allelopathic substance secreted from C. closterium. The fractions (Rt53 and Rt55) with algicidal activity were purified to chromatographic homogeneity on HPLC by sequential procedures consisting of chromatography on a reverse phase cartridge (Oasis HLB), HPLC on a gel filtration column (Superdex peptide), a reverse phase preparative column (L-column), and a reverse phase analytical column (L-column) from the non-treated medium. The 24 h LCsub(50) of Rt53 and Rt55 for H. akashiwo were 50 ng/mL and 47 ng/mL, respectively, which is similar to the 24 h LCsub(50) of ODTA (72 ng/mL).