從藥物開發與基因研究探討鐮孢菌之防治方法

Abstract

鐮孢菌 (Fusarium spp.)具病原性者影響層面廣泛，又食安問題、環境的永續性近年來備受重視，許多研究者投入非農藥資材的開發，而從巨觀至基因層次詳細瞭解病原性、抗性或藥物對病原的作用機制，將有助於更準確的對症下藥或開發新藥。 本研究共分兩部分，第一部分研究自多種天然資材中，篩選出天然資材孟寧素 (monensin)，經過最低抑菌濃度 (minimum inhibitory concentration, MIC)與最低殺菌濃度 (minimum fungicidal concentration, MFC)測試證實，孟寧素對7株來自於植物或人類之病原性鐮孢菌的MIC值最低達16 μg/mL，抑菌效果屬靜菌作用。孟寧素抑制鐮孢菌菌絲生長、影響隔膜生成，對F. oxysporum與F. graminearum之抑制效果較佳，但對F. solani則較差。由亞甲基藍染色與DAPI染色，結果顯示經孟寧素處理後之鐮孢菌細胞仍可存活，但細胞核的分離受到影響。植物病害防治方面，孟寧素較適合作為保護劑於病原入侵植物組織前或於初期感染時施用。若欲使用孟寧素針對人體病原性鐮孢菌進行治療，可以添加於眼藥水中以防範鐮孢菌於其中滋生。 第二部分研究中，對F. graminearum PH-1 (Fg)與F. oxysporum f. sp. lycopersici 4287 (Fol)建立psd2突變株以探討PSD2基因功能。PSD2基因是一磷脂質生合成相關基因，本研究探討此基因對phosphatidylethanolamine (PE)生合成、菌絲生長、產生各種孢子與致病性等能力之影響。在薄層層析試驗中可見Fg與Fol psd2突變株之PE生成量較野生株少。比較添加ethanolamine前後之菌株在微量元素培養基 (minimal medium, MM)之生長情形，發現ethanolamine可恢復Fg與Fol psd2突變株之營養缺陷。Fg與Fol之psd2突變株在MM和PDA培養基生長較野生株緩慢。在產孢方面，PSD2基因影響Fg分生孢子形態與有性世代子囊殼的形態，也影響Fol之分生孢子發芽率、厚膜孢子之產孢與其在菌絲上的著生形式。PSD2基因影響Fg胞外酵素分泌，但不影響Fg與Fol對小麥或番茄寄主之毒力。

Pathogenic Fusarium species have a broad impact worldwide. Due to the rising attention on food safety and environmental sustainability issues, we thus interested to develop non-pesticide materials. In discovering control methods through obtaining a better knowledgement of pathogenicity, drug resistance, or mechanism of drugs on pathogens from giant to gene level, we would be able to solve problem accurately or discover new drug effectively. There are two parts in this study. In the first part, monensin, a nature compound, was discovered and tested its minimum inhibitory concentration (MIC) equal or larger to 16 μg/mL against seven plant or human pathogenic Fusarium species. Inhibitory effect of monensin on Fusarium species is fungistatic. Monensin affected hyphal growth, formation of septa, and nuclear division of Fusarium species. Compared with F. solani, monensin had stronger inhibition ability against F. oxysporum, especially F. oxysporum f. sp. lycopersici 4287 (Fol) in peat extract, and F. graminearum PH-1 (Fg). For controlling Fusarium infections on plants, monensin can be recommended as protectant, and for controlling human Fusarium infections, it might be added in eye drops or contact lens solution to prevent keratitis. In the second part of study, Δpsd2 mutants of Fg and Fol were constructed and the influence of PSD2 gene, encoding phosphatidylserine decarboxylase, on phosphatidylethanolamine (a kind of phospholipid) production, hyphal growth, production of conidia or chlamydospores, and virulence in the plant hosts was tested. Comparing the growth of strains in the absence or presence of ethanolamine (Etn) in minimal medium (MM), Δpsd2 mutants of both Fg and Fol exhibited Etn auxotrophy. Δpsd2 mutants of both Fg and Fol grew slower on MM and PDA, while similar on barley bran media compared with their wild type, respectively. PSD2 gene affected conidiation and perithecia morphology of Fg. It also affected the germination rate of conidia, production and formation of chlamydospores of Fol. Although PSD2 gene affected secretion of extracellular enzymes of Fg, it is dispensable to virulence of Fg and Fol on wheat or tomato hosts.