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研究生:武藤真知子
研究生(外文):Machiko Muto
論文名稱:蘿蔔種子粉水溶性抽出物防治萵苣褐斑病的功效
論文名稱(外文):Effect of Water-soluble Extract of Radish Seed Meal on Control of Lettuce Brown Spot
指導教授:黃振文黃振文引用關係
指導教授(外文):J. W. Huang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物病理學系
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:英文
論文頁數:83
中文關鍵詞:萵苣褐斑病菌植物源農藥萵苣萵苣褐斑病藥用植物蘿蔔種子萊菔素
外文關鍵詞:Acremonium lactucumBotanical pesticideslettucelettuce brown spotmedicinal plant materialsradish seed mealsulforaphene
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近年來,台灣施行有機栽培法的農田,出現一種由Acremonium lactucum引起的萵苣褐斑病,且有逐漸蔓延的趨勢。本研究首先探討一百零三種藥用植物的水溶性抽出物抑制本病原菌的功效,結果發現十一種藥用植物之1﹪(w/v) 水溶性抽出物可顯著抑制病原菌分生孢子發芽;其中紫菀、山茱萸、山楂、萊菔子、大黃、地榆和訶子等七種藥用植物在0.25﹪的濃度下,即可完全抑制孢子發芽。將此七種藥用植物抽出物混合病原菌(105 conidia/ml)施用於株齡四個星期的萵苣植株上,結果Raphanus sativus (萊菔子)及Rheum palmatum (大黃)可顯著降低萵苣褐斑病的發病率達50﹪以上。選擇蘿蔔種子粉水溶性抽出物作為研發植物源農藥的主要成分,因此由十三種不同蘿蔔品種檢定它們的抑菌功效,結果發現赤丸金門、二十日大根、夏豐二號及晚生大梅花等品種在0.2﹪(w/v)濃度即可表現抑制病原菌發芽的效果。取蘿蔔晚生大梅花品種的水溶性抽出液(0.5﹪, w/v)在接種病原菌前三天或後兩天噴佈於萵苣植株,可有效抑制萵苣褐斑病的發生。若每星期噴佈該水溶性抽出液一次,連續三次後,發現萵苣褐斑病的罹病度可顯著的下降。將蘿蔔種子粉抽出物混合不同氮、磷、鉀、鈣及鎂化合物後,噴佈於接種過褐斑病的萵苣植株,結果顯示亞硝酸鈉、磷酸氫二鉀及醋酸鉀等三種化合物具有促進蘿蔔種子粉水溶性抽出物防治萵苣褐斑病的效果。綜合本試驗結果顯示蘿蔔種子粉抽出物中含有某些具有抑菌功效的成分,並有研發成為生物製劑的潛力,因此利用SPE及HPLC的方法分離蘿蔔種子粉抽出物中之一種有效抑菌成分,進而以UV、CI-MS、1H NMR、13C NMR及IR等方法鑑定該成分為萊菔素 (sulforaphene),是isothiocyanate群的一種。未來將研究萊菔素的抗生活性及蘿蔔種子粉內其他抑菌的成分,並嘗試探討如何將這些有效成分實際應用在生產線上,有效防治萵苣褐斑病。
A new disease of lettuce, leaf brown spot caused by Acremonium lactucum, has become a serious problem in organic farming in Taiwan during recent years. Disease control experiments were attempted by use of water-soluble extracts of 103 different medicinal plants. Eleven plant materials were effective in inhibiting conidial germination of the pathogen at 1 % (w/v) concentration on the slide culture, and among them seven which including Aster tataricus, Cornus officinalis, Crataegus pinnatifida, Raphanus sativus, Rheum palmatum, Sanguisorba officinalis, and Terminalia chebula, showed complete inhibition at 0.25% (w/v). When these plant extracts were applied respectively together with conidial suspensions (105 spores/ml) of the pathogen onto four-week-old lettuce plants, ones from Raphanus sativus (radish seed meal) and Rheum palmatum showed great efficacy of disease control by reducing the disease severity more than 50%. In this study, radish seed meal was selected as a candidate for the development of botanical pesticide formulation. A preliminary screening of the extracts from various cultivars of radish indicated that cultivars Akamaru Kinmon Hatsuka Daikon (AK-H), Hsia Heng No. 2 (HH-2), and Wan Sheng Ta Mei Hua (WS-T) were superior candidates; the extracts obtained from these cultivars inhibited completely the conidial germination of two pathogen isolates at 0.2% (w/v) concentration. In the laboratory and greenhouse trials, the effect of water-soluble extract from 0.5% (w/v) cv. WS-T radish seed meal on the disease suppression was stable when the extract was sprayed onto lettuce plants three days before inoculation or two days after inoculation. However, the disease severity of lettuce brown spot was significantly reduced when the plants were continuously sprayed three times with the extracts once per week. Seven nitrogen, five phosphate, five potassium, four calcium, three magnesium sources were mixed respectively with water-soluble extract obtained from cv. WS-T radish seed meal and tested for their effectiveness in controlling the disease. NaNO2, K2HPO4, and CH3COOK were effective in increasing the function of the extract on reducing the disease severity. These results indicated that the water-soluble extract of radish seed meal of contained certain unknown components effective in inhibiting the pathogen and thus was of great potential for developing a biocontrol formulation to protect lettuce from the disease. One of the antifungal compounds was isolated from water-soluble extract of radish seed meal with SPE and HPLC. The compound was characterized by the chemical analyses including UV, CI-Mass, 1H and 13C NMR, and IR spectrum. It was confirmed as sulforaphene, which belongs to the isothiocyanate group and derives from glucoraphenin. In future, it is necessary to determine what bio-activity of sulforaphene and other antifungal components in radish seed meal are involved in suppressing the disease development and how to apply the extract for controlling the disease in the fields.
Chinese abstract……………………………………………………………1
English abstract ……………………………………………………………3
Introduction…………………………………………………………………6
Materials and Methods……………………………………………………11
Isolates of Acremonium lactucum……………………………………11
Inoculum preparation……………………………………………………11
Plant preparation………………………………………………………11
Inoculation assay method………………………………………………12
Evaluation of disease severity………………………………………12
Preparation of water-soluble extracts from medicinal plant materials……………………………………………………………………13
Effect of water-soluble extracts from medicinal plant materials on conidial germination……………………………………13
Effect of water-soluble extracts from medicinal plant materials on disease severity…………………………………………14
Effect of water-soluble extract of radish seed meal from various cultivars on conidial germination…………………………………………………………………14
Minimum inhibitory concentration of the water-soluble extract of radish seed meal on conidial germination………………………15
Effect of water-soluble extracts of radish seed meal at different concentrations on disease severity………………………15
Effect of spraying frequency of the water-soluble extract of radish seed meal on disease severity…………………………………16
Application method of the water-soluble extract of radish seed meal for controlling the disease………………………………16
Effect of the water-soluble extract of radish seed meal with various nutrients on disease severity………………………………17
Effect of autoclaved and non-autoclaved water-soluble extract of radish seed meal on conidial germination………………………19
Evaluation of fungicidal activity of water-soluble extract from radish seed meal with different incubation periods by conidial germination assay………………………………………………19
Fungicidal activity of volatile compounds from radish seed meal to conidial germination……………………………………………20
Effect of volatile compounds from radish seed meal on inhibition of mycelial growth…………………………………………20
Isolation and determination of water-soluble antifungal compounds in the extract from radish seed meal……………………21
Statistical analysis……………………………………………………23
Results………………………………………………………………………24
Effect of water-soluble extracts from medicinal plant materials on conidial germination……………………………………24
Effect of water-soluble extracts from medicinal plant materials at different concentrations on conidial germination.24
Effect of water-soluble extracts from medicinal plant materials on disease severity…………………………………………25
Effect of water-soluble extract of radish seed meal with different concentrations on conidial germination…………………26
Effect of water-soluble extract of radish seed meal on disease severity……………………………………………………………27
Effect of spraying frequency of the water-soluble extract of radish seed meal on disease severity…………………………………28
Application method of the water-soluble extract of radish seed meal for controlling the disease………………………………28
Effect of the water-soluble extract of radish seed meal with various nutrients on disease severity………………………………29
Effect of autoclaved and non-autoclaved water-soluble extract of radish seed meal on conidial germination………………………30
Evaluation of fungicidal activity of water-soluble extract of radish seed meal with different incubation periods by conidial germination assay…………………………………………………………30
Fungicidal activities of volatile compounds from radish seed meal for conidial germination…………………………………………31
Effect of volatile compounds from radish seed meal on inhibition of mycelial growth…………………………………………31
Isolation and determination of antifungal compounds from water-soluble extract of radish seed meal…………………………32
Discussion……………………………………………………………………34
Literature cited……………………………………………………………38
Tables and Figures…………………………………………………………44
Appendix………………………………………………………………………79
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