Rapid Identification of Phytotoxins Produced by Glomerella cingulata Using High‐Resolution Mass Spectrometry‐Based Qualification, Targeted Structural Confirmation and Their Characteristics Investigation

Glomerella cingulata is a pathogenic fungus that can cause apple Glomerella leaf spot (GLS), a new and destructive apple disease in China. Phytotoxins are important factors closely related to the disease process, but there is still no report on the phytotoxins of G. cingulata. The aim of this study was to rapidly identify the phytotoxins of this pathogen using a strategy of HRMS‐based preliminary qualification, followed by targeted structure confirmation and also investigation of phytotoxicity characteristics. First, the crude toxin sample was directly analyzed by the UPLC‐HRMS and GC‐MS, and the data were processed to screen for possible phytotoxic compounds using MS library and the phytotoxicity‐related literature. The reference standards of credible phytotoxic compounds were then subjected to targeted structure validation (signal comparison between standards and compounds in crude toxin via HPLC‐DAD, UPLC‐MS/MS, and GC‐MS), and also the phytotoxicity assay. The results confirmed six phytotoxins produced by G. cingulata, namely 5‐hydroxymethyl‐2‐furancarboxylic acid (HMFCA), 2,5‐bis(hydroxymethyl)furan (BHMF), 2‐furoic acid (FA), 2,3‐butanediol, trans‐aconitic acid (TAA), and cis‐aconitic acid (CAA). Of these, HMFCA and TAA exhibited greater phytotoxicity. Main characteristics: All of them were non‐host‐selective toxins, and toxins were synergistically phytotoxic to the host when mixed. BHMF, HMFCA, FA, TAA, and CAA could be commonly produced by all tested strains, and their phytotoxicity can be significantly inhibited or even eliminated at high temperatures or high pH. The elucidation of the phytotoxins of G. cingulata in this work could provide information on the pathogenesis and control of apple GLS.