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  • 學位論文

麻豆文旦及白柚果實之流膠對柑桔潰瘍病菌(Xanthomonas citri subsp. citri)的抑菌效果及其有效成分分析

Inhibition effect of fruit gum produced by Wentan and Pai-pomelo on growth of Xanthomonas citri subsp. citri and analysis of its antibacterial components

指導教授 : 陳滄海

摘要


柑桔細菌性潰瘍病(Citrus Bacterial Canker)是由Xanthomonas citri subsp. citri引起,能夠危害柑桔類作物之葉片、枝條及果實,是國際柑桔栽培上最重要且具破壞性之細菌性病害。柑桔類果樹為台灣重要經濟果樹,其中文旦及白柚屬柑桔潰瘍病耐病品種,但以往調查結果顯示,文旦及白柚葉片因颱風侵襲受傷後,嚴重遭受潰瘍病危害。本研究採集田間不同品種之柑桔類罹病組織,經分離及鑑定後,確認罹病組織分離之潰瘍病菌均屬亞洲型潰瘍病菌。在調查期間,田間果實僅少數遭受潰瘍病之危害,且發現受傷或經人工穿刺處理後之果實,於果實傷口處會有流膠現象產生。進一步檢測果實流膠之功能發現,濃度20,000 ppm之文旦與白柚果實流膠對同屬之柑桔潰瘍病菌、十字花科黑腐病菌(X. campestris pv. campestris)、茄科細菌性斑點病菌(X. axonopodis pv. vesicatoria)及水稻白葉枯病菌(X. oryzae pv. oryzae)之生長皆有明顯抑制效果。為確認果實流膠與果實抗潰瘍病之關聯性,於2008年3至8月文旦及白柚結果期間,採集田間不同齡期之果實,經人工穿刺製造傷口 後,傷口處皆可發現產生流膠現象。進一步檢測不同結果期果實流膠對柑桔潰瘍病菌之抑菌效果差異顯示,在20,000 ppm濃度下,2008年結果期3至7月之台東文旦果實流膠,對受檢測潰瘍菌株平均生長抑制圈在2.81±1.45至10.45±2.23 mm;結果期3至8月之台南文旦果實流膠,對受檢測潰瘍菌株平均生長抑制圈在0.78±0.65至16.5±2.19 mm;結果期4至8月之台南白柚果實流膠,對受檢測潰瘍菌株平均生長抑制圈在0.00±0.00至16.83±1.53 mm;除結果期5月之台南文旦果實流膠與結果期8月之台南白柚果實流膠外,其餘月份果實流膠對柑桔潰瘍病菌皆有明顯抑菌效果。將健康株果實、罹病株罹病果實及罹病株無病徵果實經人工穿刺後,發現傷口處均會產生流膠,且罹病株罹病果實流膠對果實分離之潰瘍病菌菌株仍具抑菌效果。上述三種果實流膠在20,000 ppm濃度下分別對檢測菌株有22.9%、52.1%及14.6%之抑菌率。果實流膠特性檢測結果顯示,以9.09 mg ml-1之蛋白質酵素K處理後,抑菌能力會喪失,顯示抑菌物質可能為蛋白質分子;水為最佳萃取溶劑;果實流膠在酸鹼值2至11的範圍內,皆具有抑菌活性;文旦果實流膠通過DEAE Sepharose陰離子交換樹脂管柱後,獲得一個陽離子、三個陰離子層析樣本,其中以CF1 fraction樣本在濃度為1 g/ml時之抑菌效果較佳,平均生長抑制圈為12.5±2.07 mm。再利用SepharoseTM CL-6B膠體管柱將CF1 fraction成分分離,獲得CF1-1及CF1-2兩個fractions,在濃度為1 g/ml時平均生長抑制圈分別為9.67±0.82及10.00±0.63 mm,經Tukey統計分析無顯著差異;但觀察軟瓊脂平板,發現CF1-2 fraction抑制圈內菌落生長並未完全受到抑制。經SDS-PAGE分析,得知CF1-1 fraction具有一個約30 kDa之蛋白質分子,推斷此一蛋白質分子為果實流膠抑菌主要有效成分之一。CF1-1 fraction經質譜分析及MASCOT資料庫蛋白質比對,結果顯示其與一23 kDa的蛋白質GL22_ORYSJ (Accession NO. Q6K5P9)序列有7%相同度,比對到一段16個胺基酸序列,推測文旦果實流膠CF1-1 fraction之抑菌成份可能與類發芽素蛋白質(germin-like protein, GLPs)有關或為其成員之一。

並列摘要


Citrus bacterial canker caused by Xanthomonas citri subsp. citri (Xac) could cause damage to leaves, branches and fruits of citrus. It was the severest and destructive bacterial disease on the international citrus cultivation. Citrus was a very important commercial fruit trees in Taiwan, Wentan-pomelo and Pai-pomelo were tolerant varieties to citrus bacterial canker. But previous study indicated that Wentan-pomelo and Pai-pomelo leaves were infected by citrus bacterial canker after typhoon invasion. The Asiatic canker pathogen were isolated from infected tissues of different citrus cultivars, and identified by PCR. During the investigation, it was found that the fruits of Wentan-pomelo and Pai-pomelo were scarcely infected by citrus bacterial canker, and in the meantime the gum was found around the wound that caused by injury or puncture treatment on fruits. The inhibition effects of the fruit gum solution of Wentan-pomelo and Pai-pomelo to X. citri subsp. citri, X. campestris pv. campestris, X. axonopzodis pv. vesicatoria and X. oryzae pv. oryzae were observed in the concentration 20,000 ppm. In order to confirm the relationship between the fruit gum and fruit resistance to citrus bacterial canker, the fruit gum collected from different age fruits of Wentan-pomelo and Pai-pomelo from Taitung and Tainan in March to August 2008, were tested and shared the inhibition effect to citrus bacterial canker pathogen (Xac) with the concentration of 20,000 ppm obviously. All the healthy, Xac-infected and symptomless fruits collected from diseased Wentan-pomelo plant could produce the gum around wound after puncture treatment, and also showed the inhibition effect to citrus bacterial canker pathogen (Xac) with 22.9%, 52.1% and 14.6% respectively. The previous result showing the fruit infection by citrus bacterial canker was not due to the loss of fruit gum producing ability or the resistance to fruit gum by citrus bacterial canker pathogen (Xac). The fruit gum was inactive after treatment with proteinase K, indicated that the antimicrobial component might be a protein. Water was the best extraction solvent for fruit gum, and the inhibition activity to Xac was still best worked under pH2 – pH11. One cation (CF1) and three anion (AF1, AF2 and AF3) fractions were isolated by DEAE sepharose ion exchange chromatography from the fruit gum of Wentan-pomelo, and the CF1 fraction sample showed the best inhibition effect with 12.5±2.07 mm inhibition range at concentration of 1 g/ml. Two fraction, CF1-1 and CF1-2, were isolated from CF1 fraction by SepharoseTM CL-6B gel filtration chromatography, and showed the inhibition range of 9.67±0.82 and 10.00±0.63 mm respectively. Though it was not significantly different in Tukey test; but the colonies were not completely inhibited by CF1-2 on soft agar plates. One component with 30 kDa in molecular weight was detected in CF1-1 by SDS-PAGE analysis, and the component could be a protein and was possibly the major active antimicrobial component of fruit gum. The protein GL22_ORYSJ (Accession NO. Q6K5P9) matched with the fruit gum antimicrobial protein that showed 7% identity of amino acid sequence and an amino acids sequence with 16 aa was confirmed by mass spectrometer analysis and protein database comparison. In conclusion the antimicrobial protein of fruit gum was possibly related to or could be a member of germin-like protein.

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