草莓炭疽病菌之毒性代謝物鑑定與功能性分析

Abstract

草莓為台灣重要的高經濟作物，炭疽病菌可潛伏感染於草莓種苗中，至本田期大規模發病，造成嚴重危害。前人研究發現，辣椒及菸草炭疽病菌分泌之毒性代謝物屬小分子化合物，接種於寄主會呈現類似病原菌感染之病徵，但目前對於炭疽病菌毒性代謝物之化學結構，及毒性代謝物在侵染寄主過程中扮演之角色均有待釐清。本研究欲探討草莓炭疽病菌之毒性代謝物種類與功能，首先建立草莓葉圓葉片生物測定法 (leaf disc bioassay)，同時以加入草莓葉汁液等方法調整液態培養條件，增加毒素產量，發現培養過草莓炭疽病菌之濾液可在離葉與圓葉片產生和病原菌相似的壞疽病徵。為瞭解毒性代謝物之相關特性，將濾液接種於草莓、菸草、木瓜、青江菜、小麥、香蕉與水稻等不同單子葉與雙子葉植物之圓葉片，確認毒性代謝物不具寄主專一性。另將濾液以高溫高壓滅菌和蛋白質酶處理後，仍無法抑制其誘導產生壞疽病徵之活性，確認活性物質具耐熱性且並非蛋白質。透過Diaion HP20 open column chromatography純化毒性代謝物，再配合高效液相色譜法 (high performance liquid chromatography)、質譜法 (mass spectrometry) 與核磁共振 (nuclear magnetic resonance) 等分析技術，鑑定活性物質為2,5-bis(hydroxymethyl)furan (BHMF)；購買商品化之BHMF接種草莓葉圓片與離葉，確實可產生類似炭疽病菌感染之病徵。本研究亦透過農桿菌轉殖法，篩選出一株毒性代謝物缺陷型突變株 (BHMF產量較野生型低~36%)，將其接種於草莓離葉後，造成的病斑比野生型顯著較小，顯示BHMF可能是草莓炭疽病菌的毒力因子。至於BHMF在草莓葉片或冠部組織之作用濃度，以及是否有其他因子協力作用等，則有待進一步釐清。

Strawberry is a high value fruit crop in Taiwan. Latent infected of Colletotrichum gloeosporioides in strawberry seedlings can cause severe damage to field-grown plants. In previous studies, the toxic metabolite(s) of Colletotrichum sp. were identified as low molecular weight substances; inoculation of the culture filtrates led to lesions similar to that caused by pathogen infections. However, for Colletotrichum spp., precise chemical structures of the toxic metabolite(s) and the roles of toxic metabolite(s) in pathogenesis remain to be clarified. To investigate the mechanism underlying pathogenesis of C. gloeosporioides, this research focused on the identification and functional characterization of toxic metabolite(s) produced by C. gloeosporioides. We optimized toxic metabolite(s) production by supplementing the medium with strawberry leaf extract and developed a leaf disc bioassay method for phytotoxic activity evaluation. The culture filtrate of C. gloeosporioides 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, including strawberry, tobacco, papaya, bok choy, wheat, banana, and rice suggested host nonspecificity of the toxic metabolite(s). Autoclaving or pretreatment with 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. By employing Diaion HP20 open column chromatography, RP-amide high performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance, the toxic metabolite was isolated and identified as 2,5-bis(hydroxymethyl)furan (BHMF). Commercially available BHMF was able to induce anthracnose-like symptoms on detached leaves and leaf discs of strawberry. A toxin-deficient mutant which produced ~36% less BHMF than the wild type was generated by using Agrobacterium-mediated transformation. Inoculating the toxin-deficient mutant on detached strawberry leaves resulted in significantly smaller lesions than the wild type, suggesting that BHMF is a virulence factor of C. gloeosporioides in strawberry. Future work will focus on investigating the effective concentration of BHMF in strawberry leaf and crown tissues, as well as understanding whether there is synergistic factor(s) of BHMF.