臺灣博碩士論文加值系統

Fusarium oxysporum f. sp. niveum (Fon) 引起之西瓜蔓割病為台灣西瓜栽培的主要限制因子之一。目前抗病育種乃防治西瓜蔓割病之最佳策略。前人研究中發現在抗病西瓜品系 JSB 之莖基部上苯丙氨酸氨裂解酶 (phenylalanine ammonia lyase, PAL) 之基因表現可能和西瓜抗蔓割病有關，而 PAL 為苯基丙酸代謝路徑之第一步調控酵素，其下游產物酚化物 (phenolic compounds) 在植物中提供多種抗病機制包括：訊息分子、抗微生物物質及結構抗性聚合物。為了探討西瓜抗蔓割病的機制，本試驗以西瓜抗病品系 JSB 為研究對象並以感病西瓜品種藍寶 (Grand Baby, GB) 做為對照組，將西瓜幼苗種植於帶菌病土中，觀察其病勢發展與接種後植株內病原菌的纏據，並分析接種後 3、 6、 9、 12 及 15天西瓜苗各組織中 PAL 的酵素活性與分析苯基丙酸代謝路徑下游各類酚化物質包括可溶性酚化物以及與細胞壁結合酚化物，並進一步分析可溶性酚化物中的未鍵結酚化物、酯類鍵結酚化物和醣基鍵結酚化物。同時利用組織切片染色的方法觀察西瓜莖基部維管束之木質素含量，以及分析與西瓜幼苗酚化物氧化相關的過氧化酶 (peroxidase, POD) 及多酚氧化酶 (polyphenoloxidase, PPO) 在接種過後的酵素活性。結果顯示雖然抗病品系 JSB 接種後無病徵顯現，但仍然能在莖基部與下胚軸組織中分離出西瓜蔓割病菌。而抗病品系 JSB 在莖基部之 PAL酵素在接種過後明顯提高其活性，而在酚化物含量上，抗、感病西瓜苗在接種後下胚軸及莖基部可溶性酚化物及細胞壁鍵結酚化物在含量上皆明顯提高，然而感病植株在接種後期可溶性酚化物及細胞壁鍵結酚化物含量呈現下降的趨勢，而利用化學方法分析可溶性酚化物之成分，發現抗病植株莖基部及根部之酯類鍵結酚化物在接種過後其含量較高，同時切片染色觀察也發現抗病品系 JSB 莖基部維管束在接種過後有明顯木質素累積現象，且在接種後第 6 天發現在抗病西瓜品系 JSB 根部及莖基部之過氧化酶活性明顯提高。總結這些結果顯示抗病西瓜品系 JSB 在接觸病原之根部與莖基部上，由於酯類鍵結酚化物質含量之提高，加上過氧化酶活性的提前增高，使得木質素快速的在抗病西瓜品系 JSB 的維管束中累積，因而提供抗病品系 JSB 有效的結構抗性。

Fusarium wilt of watermelon, caused by Fusarium oxysporum f. sp. niveum (Fon), is one of the limiting factors on watermelon production in Taiwan. Breeding for disease-resistant watermelon is the best strategy to control Fusarium wilt. In recent research, the gene expression of phenylalanine ammonia lyase (PAL) in shoot base of a Fusarium wilt resistant line (JSB) was related to Fusarium wilt resistance. PAL is the first step regulatory enzyme in phenylpropanoid metabolic pathway. The downstream products are phenolic compounds which provide many functions in plant resistance such as signal molecules, antimicrobial substance and structure barriers. To study the resistant mechanism of Fusarium wilt, a Fusarium wilt resistant JSB line was used to compare with a Fusarium wilt susceptible Grand Baby (GB) cultivar after Fon inoculation. The resistant and susceptible watermelons were grown in Fon-infested soil, the disease progress and pathogen colonization in plant were observed. We then analyzed the enzyme activity of PAL and the amounts of soluble and cell wall-bound phenolic compounds in different parts of watermelon seedling at 3, 6, 9, 12, and 15 days after inoculated with Fon. Different kind of phenolics, i.e. free phenolics, ester-bound phenolics, and glycoside-bound phenolics, were also analyzed. Lignin deposition in shoot base tissue weas also observed by histochemistry. The activities of two phenol oxidative enzymes, peroxidase (POD) and polyphenoloxidase (PPO) were further analyzed. The results showed that although the resistant JSB line showed no symptom after inoculation, Fon pathogen could still be recovered from shoot base and hypocotyl tissues. Significant increase in PAL activity was found in the shoot bases of resistant JSB line after inoculation. Both resistant and susceptible watermelons have significant increase in the amounts of soluble and cell wall-bound phenolics, the susceptible GB cultivar, however, has the tendency of decrease in the amount of phenolics afterward. The amount of ester-bound phenolic compounds was determined chemically and significant increase was found in resistant JSB line after Fon inoculation. In histochemistry study, lignin deposition in the cell wall of vascular bundle was also observed in the shoot bases of JSB seedlings. In the roots and shoot bases of JSB seedlings at 6 days post Fon inoculation, POD enzyme activity increased significantly. In summary, our results suggest that ester-bound phenolics and POD activity in roots and shoot bases of JSB seedlings after Fon inoculation, together with the rapid deposition of lignin in the cell wall of vascular bundle may provided the structure barrier in resistant JSB line to defend Fon invasion

目次
中文摘要…………………………………………………………………………….　.　i
英文摘要…………………………………………………………………………….ii
前言…………………………………………………………………………………...1
材料與方法………………………………………………………………………….5
供試菌株來源與病土製作………………………………………………………5
供試植株及接種…………………………………………………………………5
組織分菌…………………………………………………………………………5
苯丙氨酸氨裂解酶酵素活性……………………………………………………6
酚化物之萃取及分析……………………………………………………………6
(一)可溶性酚化物之萃取.................................................................................6
(二)未鍵結酚化物之萃取................................................................................7
(三)酯類鍵結酚化物之萃取.............................................................................7
(四)醣基鍵結酚化物之萃取.............................................................................7
(五)細胞壁鍵結酚化物之萃取.........................................................................7
西瓜莖基部之組織化學觀察……………………………………………………8
酚化物氧化相關酵素活性…………………………………………………8
(一)酵素之萃取…………………………………………………………….....8
(二)過氧化酶酵素活性測定……………………..………………………….9
(三)多酚氧化酶酵素活性測定…………………………..………………….9
結果………………………………………………………………………………….10
病原菌之纏據及 PAL 之酵素活性…………….……………………………..10
(一)組織分菌……………………………………………………….………..10
(二)苯丙氨酸氨裂解酶酵素活性………………………….………………..10
各類酚化物在接種過後於各組織中含量之差異………………………...…...11
(一)植株整體酚化物含量差異……………………………………………11
(二)各類酚化物於子葉之含量差異……………………..………………..11
(三)各類酚化物於下胚軸之含量差異……………………...………………12
(四)各類酚化物於莖基部之含量差異……………………………….……..12
(五)各類酚化物於根部之含量差異………………………………………..12
抗病植株結構抗性之觀察………………………………….………………….13
(一)西瓜莖基部組織化學觀察……….……………………………………..13
(二)過氧化酶及多酚氧化酶酵素活性….…………………………………..13
討論………………………………………………………………………………….15
參考文獻…………………………………………………………………………...19
圖表………………………………………………………………………………….23
附圖一………………………………………………………………………………41
附圖二........................................................................................................................42
附表.............................................................................................................................43

















圖表目次
圖一、抗、感病西瓜幼苗接種西瓜蔓割病菌 (Fon-H0103菌株) 後各組織內之帶菌百分比...........................................................................................................23
圖二、抗、感病西瓜幼苗接種西瓜蔓割病菌 (Fon-H0103菌株) 後各組織中苯丙氨酸氨裂解酶 (Phenylalanine ammonia lyase，PAL) 的酵素活性.......................................................................................................................25
圖三、抗、感病西瓜幼苗接種西瓜蔓割病菌 (Fon-H0103菌株) 後各植株整體可溶性酚化物及細胞壁鍵結酚化物含量...........................................................27
圖四、抗、感病西瓜幼苗接種西瓜蔓割病菌 (Fon-H0103菌株) 後子葉之可溶性酚化物及細胞壁鍵結酚化物含量...................................................................28
圖五、抗、感病西瓜幼苗接種西瓜蔓割病菌 (Fon-H0103菌株) 後子葉之未鍵結、酯類鍵結及醣基鍵結之酚化物含量...............................................................29
圖六、抗、感病西瓜幼苗接種西瓜蔓割病菌 (Fon-H0103菌株) 後下胚軸之可溶性酚化物及細胞壁鍵結酚化物含量...............................................................30
圖七、抗、感病西瓜幼苗接種西瓜蔓割病菌 (Fon-H0103菌株) 後下胚軸之未鍵結、酯類鍵結及醣基鍵結之酚化物含量.........................................................31
圖八、抗、感病西瓜幼苗接種西瓜蔓割病菌 (Fon-H0103菌株) 後莖基部之可溶性酚化物及細胞壁鍵結酚化物含量...............................................................32
圖九、抗、感病西瓜幼苗接種西瓜蔓割病菌 (Fon-H0103菌株) 後莖基部之未鍵結、酯類鍵結及醣基鍵結之酚化物含量.........................................................33
圖十、抗、感病西瓜幼苗接種西瓜蔓割病菌 (Fon-H0103菌株) 後根部之可溶性酚化物及細胞壁鍵結酚化物含量...................................................................34
圖十一、抗、感病西瓜幼苗接種西瓜蔓割病菌 (Fon-H0103菌株) 後根部之未鍵結、酯類鍵結及醣基鍵結之酚化物含量.........................................................35
圖十二、抗、感病西瓜幼苗於接種 Fon 過後其莖基部維管束中木質素的累積情形.......................................................................................................................36
圖十三、抗、感病西瓜幼苗接種西瓜蔓割病菌 (Fon-H0103菌株) 後各組織中過氧化酶 (Peroxidase，POD) 的酵素活性.........................................................37
圖十四、抗、感病西瓜幼苗接種西瓜蔓割病菌 (Fon-H0103菌株) 後各組織中多酚氧化酶 (Polyphenoloxidase，PPO) 的酵素活性.........................................39

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