三唑類殺菌劑對炭疽病菌 Colletotrichum spp. 之鏡像選擇性研究

Abstract

本研究利用液相層析的鏡像異構物分離技術，將鏡像異構物分離後，比較其抑菌能力的差異，盼以利用有效異構物，排除無效異構物的方式降低三唑類 (triazole) 殺菌劑之使用量。研究標的選擇待克利 (difenoconazole)、達克利 (diniconazole)、菲克利 (hexaconazole)、普克利 (propiconazole) 及得克利 (tebuconazole) 等具高用藥頻度的農藥為研究對象，並將炭疽病病原真菌 Colletotrichum spp. 作為受試生物以量化單離之鏡像異構物有效性。三株病原真菌 GLA, BY2 和 Ing01 透過組織分離法，自疑似感染炭疽病果實表面分離，各菌株以聚合酶連鎖反應增幅特定序列，並與 NCBI 資料庫相比對完成所分離菌株之初步鑑定。結果顯示，GLA, BY2 和 Ing01 分別為 C. siamense, C. musae 和 C. gloeosporioides。與高效液相層析儀配合之下，鏡像管柱 Chiralcel OJ-H 能對達克利、菲克利和得克利的鏡像異構物分離具有高解析度，分別為 14.4, 4.97 和 3.11。後續以氣相層析儀串聯質譜儀分析單一鏡像異構物，分析結果與 NIST 資料庫相比對，顯示所有分離的鏡像異構物均為該原農藥，再使用圓二色光譜確認其光學活性。C. musae BY2 生物性試驗結果顯示，於濃度 1.00 mg．L-1 處理之下，(-)-R-得克利的菌絲抑制率為 94.3%，其 EC50 為 0.133 mg．L-1，藥效相當於消旋體所造成的影響，且因 (+)-S-得克利於統計分析上無顯著抑制能力，說明 (-)-R-得克利為生物試驗中主要作用型態。菲克利的兩種鏡像異構物對 C. musae BY2 均具有抑制能力，於濃度為 1 mg．L-1 的處理下，(-)-菲克利及 (+)-菲克利菌絲抑制率分別為 70.4% 及 44.9%。C. gloeosporioides Ing01生物性試驗結果顯示，兩種農藥之鏡像異構物均以 (-)-型態具有較高的抑制能力: (-)-R-得克利之菌絲抑制率為 80.9%，(+)-S-得克利為 47.5% (濃度為 1.00 mg．L-1)；(-)-菲克利之菌絲抑制率為 83.6%，(+)-菲克利則為 53.4% (濃度為 10.0 mg．L-1)。(-)-R-得克利於病原真菌 C. musae BY2 有高度的鏡像選擇性，推測原因為其構型能有效與酵素接合位結合，進而阻斷麥角固醇合成途徑，產生菌絲抑制現象。

The purpose of this study was to reduce the usage of triazole pesticides frequently used in Taiwan by enantiomer separation and subsequent test for effect on fungi Colletotrichum spp., which causes severe losses on produces annually. Results of DNA sequence analysis indicated that three isolated pathogens strain BY2, GLA and Ing01 were confirmed to be Colletotrichum spp. by comparing the conserved sequences Internal Transcribed Spacer (ITS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) through DNA sequencing analysis. The separation of enantiomers was conducted by High Performance Liquid Chromatography (HPLC). Under normal phase condition, the chiral column Chiralcel OJ-H performed great resolution of the enantiomers of Diniconazole, Hexaconazole, and Tebuconazole with resolutions of 14.4, 4.97, and 3.11, respectively. Moreover, all separated enantiomers were confirmed to be the original pesticides via Gas Chromatography-Mass spectrometry (GCMS) through specific m/z profile comparison with NIST database；the optical behavior of separated enantiomers was determined by Circular Dichroism (CD) spectra. By observing the inhibition rate against C. musae and EC50 value, (-)-R-tebuconazole is the sole effective isomer by inhibiting 94.3% of C. musae BY2’s mycelial growth, with an EC50 value of 0.133 mg．L-1, which is equivalent to the effect caused by rac-tebuconazole. In addition, both isomers of hexaconazole inhibited C. musae BY2’s mycelial growth, with 70.4% and 44.9% inhibition rate caused by (+)-hexaconazole and (-)-hexaconazole respectively. Additionally, results of inhibition tests against C. gloeosporioides Ing01 showed that (-)-R-tebuconazole exhibited greater inhibiting activity with 80.9% compared to (+)-S-tebuconazole (47.5%), similarly, (-)-isomer of hexaconazole also exhibited greater inhibition rate of 83.6% compared to (S)-isomer with 53.4%. In summary, the tested triazoles in (-)-form all performed inhibiting activity towards to Colletotrichum spp., while only the inhibition test of tebuconazole against susceptible strain BY2 exhibited excellent enantioselectivity. The difference in inhibiting ability of tebuconazole implied that the structure of enzymatic target may exhibit selectivity towards the tebuconazole forms, possibly due to highly specific request for conformation.