Identification and characterization of the potential nematicidal agent from endoparasitic nematophagous fungus, Esteya vermicola

Abstract

The pinewood nematode, Bursaphelenchus xylophilus, which is the causal agent of pine wilt disease, has become a serious threat to forest ecosystem. To control these disastrous pine wilt disease, chemical pesticides such as abamectin and emamectin benzoate had been introduced to be used as nematicides. However, there has been concern about these pesticides, which could pose side effects as they have lethal activities against a broad range of hosts. Efficient and environmentally beneficial alternatives to existing pesticides, which do not affect non-target organisms, are therefore urgently required. For this purpose, natural enemies would be an excellent source model for discovering novel agents. Esteya vermicola was reported as a first endoparasitic nematophagous fungus that attacks the pine wood nematode. Although the infection activities of E. vermicola had been verified as a biocontrol agent of pine wilt disease, how they attack pathogenic nematodes are still not fully understood. Therefore, it would be valuable to find out the biologically active agents from this natural systems followed by clarification of the underlying infection mechanism. In this study, I identified the potential virulences gene, EVERM_004614 involved in infection process through genome and transcriptome analysis of E. vermicola from nematode-fungi infection model. For functional analysis, knockout and overexpression strains of EVERM_004614 were constructed by Agrobacterium tumefaciens mediated homologous recombination. Comparative infection assay conducted with knockout and wildtype strains against B. xylophilus reavealed that EVERM_004614 plays a highly significant role in infection process. For purification and structural elucidation of compound biosynthesized from EVERM_004614, culture condition and extraction solvents were tested to obtain sufficient amount of target compounds. Furthermore, the retention time and absorption characteristic of four desired compounds were determined based on the comparative analysis of HPLC profile between wildtype and mutant strains. This work would pave a way to find environmentally beneficial alternatives, with unprecedented approaches which explore the nematicidal agent from the natural enemy of pine wood nematode.