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Identification and functional characterization of the toxin produced by Colletotrichum fragaricola in strawberry

Shiang-Shiuan Yu: Department of Plant Pathology and Microbiology, National Taiwan University

<div>Strawberry production in Taiwan has severely suffered from anthracnose, which is caused by three <em>Colletotrichum</em> species including <em>C. karstii</em>, <em>C. fructicola</em>, and <em>C. fragaricola</em> sp. nov. (a potentially new and dominant species). To investigate the mechanism underlying pathogenesis of <em>C. fragaricola</em>, this research focuses on the identification and functional characterization of toxins produced by <em>C. fragaricola</em>. We optimized toxin production by supplementing the medium with strawberry leaf extract and developed a leaf disc bioassay method for toxin evaluation. The culture filtrate of<em> C. fragaricola</em> caused necrosis on leaf discs and induced anthracnose symptoms on detached leaves of strawberry. Assays on the leaf discs of several dicot and monocot crops suggested the lack of host specificity. Pretreatment with heat or proteinase K did not inhibit the necrosis-inducing activity of the culture filtrate, indicating that the active ingredient is a thermostable and non-protein substance. We then isolated and identified the water-soluble toxic chemical(s) by employing Diaion HP20 open column chromatography, high performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance. To uncover the role of the toxin in pathogenesis, <em>Agrobacterium-</em>mediated transformation and mung bean radicle growth bioassay method were used to screen for toxin-deficient mutants. The results will provide new insights into the mechanisms of <em>C. fragaricola</em> infection in strawberry.</div>