Identification of Mellein as a Pathogenic Substance of Botryosphaeria dothidea by UPLC-MS/MS Analysis and Phytotoxic Bioassay

Botryosphaeria dothidea is a pathogenic fungus that can cause apple ring rot, a destructive apple disease in China. There have been reports on its molecular pathogenesis, but the pathogenic substances still remain unknown. In the present study, instrument analysis including UPLC-high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance showed that B. dothidea fermentation broth contained (R)-(-)-mellein, a well-known fungal enantiomer of mellein. For further confirmation, a UPLC-MS/MS method for the determination of mellein was developed and validated. By this method, mellein was found to also exist in B. dothidea-infected apple fruits and branches with concentration ranges of 0.14-0.94 and 5.88-80.29 mg/kg, respectively. The concentration in fruits reached a peak at 48 h after pathogen inoculation, while a sustained concentration increase was achieved within 11 days for branches. Simultaneously, it was evident that there was a relation between disease spot expansion and mellein production kinetics in apple tissue. Phytotoxic bioassay showed that mellein could cause discoloration and death of apple leaves and browning in stems. Therefore, we confirmed that mellein was one of the pathogenic substances of B. dothidea. The present study provided additional data for the research on the pathogenesis of this pathogen.