利用薄膜培養基法檢測褐根病菌分泌之生體外分解酵素

Abstract

褐根病為近年來台灣普遍發生的木本植物根部病害之一，被為害植物的根系組織嚴重腐敗。本文利用薄膜培養基測試法探討褐根病菌分泌10 種細胞外泌酵素 (extracellular enzymes) 之能力。試驗結果顯示分別分離自南投縣、高雄市等地區所採集罹病植株之5 支褐根病菌供試菌株 (PN18.1：分離自南投縣中興新村之櫸木；PN22.1：南投縣國姓鄉之木麻黃；PNG1.1：南投縣水里鄉之葡萄；PNP1.1：高雄市甲仙區之梅樹；及PNZ1.1：高雄市阿蓮區之印度棗) 皆能分泌澱粉分解酵素 (amylase)、纖維分解酵素 (cellulase)、果膠分解酵素 [pectinases, 含polygalacturonases (PGs) 與pectate transeliminases (PTEs)]、蛋白分解酵素 (protease)、磷酸分解酵素 (phosphatase)、尿素分解酵素 (urease)、脂質分解酵素 (lipase) 及木質分解酵素 (laccase)；但不能分泌磷脂分解酵素 (phosphatidase) 與幾丁質分解酵素 (chitinase)。除木質分解酵素外，各菌株分泌各種分解酵素的能力隨酵素種類的不同而有強弱之差異，可達到5% 顯著性水準。然而所有供試菌株分泌木質分解酵素的活性均甚高。本研究所建立褐根病菌分解酵素之檢測技術，可進一步應用於褐根病菌致病機制之研究。 Brown root rot caused by Phellinus noxius is one of the most serious diseases associated with decline of fruit trees and ornamental woody trees in Taiwan. The objective of this study was to develop a modified polycarbonate membrane method for detection of extracellular enzymes produced by P. noxius growing on solid agar media. Five isolates of P. noxius collected from different host plants in Taiwan were used in this study, including isolate PN22.1 from ironwood (Casuarina equisetifolia) at Guosing, Nantou, isolate PNG1.1 from grapevine (Vitis vinifera) at Shueili, Nantou, isolate PN18.1 from zelkova (Zelkova serrata var. serrat) at Chungsin, Nantou, isolate PNP1.1 from plum (Prunus mume) at Jiasian, Kaohsiung and isolate PNZ1.1 from Indian jujube (Zizyphus mauritiana) at Alian, Kaohsiung. They were tested for production of extracellular enzymes by growing each isolate on polycarbonate membrane disc (0.2 μm in pore diam.) which was placed on PDA amended with enzyme-reaction chemicals for detection of enzymes. Results showed that eight extracelluar enzymes, including amylase, cellulase, pectinases (polygalacturonase and pectate transeliminase), protease, phosphatase, lipase and laccase, were detected in all the five isolates of P. noxius, but no phosphatidase and chitinase were detected from these isolates. Among the eight enzymes detected, the level of laccase secreation was high in all the five isolates, but the level of secretion of other seven enzymes varied with the isolates tested. The technique for detection of enzymes can be applied to pathogenecity analysis in P. noxius and other fungi.