Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31696
標題: MR配方連續施用下對甜瓜之可能生理影響研究
Studies on the Possible Physiological Effects of Continued Application of MR Formulation on Muskmelon(Cucumis Meloncv. Non-Fa No.38)
作者: 許富翔
Hsu, Fu-Shiang
關鍵字: 甜瓜
MR配方
出版社: 植物病理學研究所
摘要: MR(甲硫胺酸與核黃素)配方之研究發展,為世界上首次將由食品營養成份所產 生的光動效應實際應用於田間植物病害防除上獲得成功的實例,其最大特色在於對環 境不致有殘毒之虞。有關MR對多數微生物的殺生作用最早係在1984年由Tzeng氏及 DeVay氏所發現,經過十年來的研究,在有關MR分子殺生作用機制之瞭解以及配方 實用性之開發上已有相當成果,最近於臺灣氰胺公司的努力下,正式化的商品即將在 今年上市,推薦推廣於瓜類白粉病之防治。然者,許多在應用上必須齊備之基本研究 資料,確還有許多不完整而有待加強研究瞭解之處,如施用後對植物之生理影響及其 棲息生態系統之影響等。基於此,本研究之目的,係以農發38號(Cucumis melon cv. Non-Fa No. 38)甜瓜為測試材料,探討MR在連續施用下對其葉部之可能生理影響。雖 然於先前試驗中顯示,MR施用不會導致供試植物藥害的產生,但試驗之初均見每次 MR施用後,乙烯與游離態脯胺酸瞬時性之產生,顯示MR對供試植物或有類似逆境 之影響,但就其光合作用相關因子,葉片細胞膜功能,以及老化作用指標等之測定, 結果發現MR處理後均對供試植物無明顯不良之影響。另就可溶性蛋白之組成,發現 其大部份蛋白組成均與噴水對照組間相同,唯於90 kDa處蛋白有瞬時性生合成之增 加。此外亦嘗試以HPLC分析游離態胺基酸中,常見14種必須胺基酸之含量,結果發 現glycine在MR處理後於葉部會有瞬時性累積的情形,此胺基酸之含量變化與MR處 理供試植物生理之變化影響究竟為何,則有待闡明。按上述之結果,比較MR處理植 株與噴水對照植株間之各項生理功能,顯示對作物之生長、發育應不致有明顯不利之 影響。由於MR殺生作用機制在於其活化態氧的產生,但就活化態氧對於生物細胞影 響之角度來看,施用MR後其所產生之活化態氧似乎對供試植物並無明顯傷害性之影 響,顯示供試植物體或有活化態氧保護機制存在,因此為探究供試植物如何在MR施 用下而不受影響。本實驗就目前已知與活化態氧清除相關酵素及非酵素因子進行測試, 其結果發現Ascorbate peroxidase、glutathione reductase、與Superoxide dismutase等酵 素之活性,於MR處理後並無明顯影響;而Peroxidase之活性則有明顯增加,此些供 試植株萃取所得之Peroxidase製備,經由等電點焦集電泳分析發現,主要增加成份為 pI值10.0左右之一同功酵素,其它大部份peroxidase同功酵素之活性則似乎無明顯變 化;與peoxidase相反的,於MR處理植株,其catalase酵素活性在處理後有很短暫的 減少現象,此一現象一般在施藥三天後即可見有恢復之趨勢,唯於利用Native PAGE 分析酵素特性時卻發現,此些回復具活性之catalase,其在電泳時之移動性質(mobility) 與對照組間有相當明顯之差異,顯示此些catalase,其蛋白分子或已有某些結構上的改 變(modification);除上述酵素種類外,於MR處理植株,其還原態glutathione與carotenoid 含量與噴水之對照植株並無明顯差別,唯氧化態glutathione與ascorbate含量於MR施 用後明顯少於噴水處理之對照植株。綜合這些與活化態氧清除有關酵素與酵素因子之 變化,不難看出MR在處理後,其所產生之活化態氧確實導致植物體與活化態氧去除 相關因子之暫時性改變。然者,除peroxidase於連續MR施用期間活性持續高於噴水 之對照植株外,其餘與活化態氧清除之相關因子中,均僅見有立即短暫之影響,顯示 MR產生之活化態氧可能為植物既存活化態氧去除機制所清除。但值得注意的是 peroxidase與catalase活性的改變與甫近常見報導salicylate所誘導產生的系統性抗病性 (SAR),似有部份雷同之處,此是否顯示MR處理後會有促進抗病性之現象很值得繼 續研究瞭解。但就本研究中現有生理影響之測試結果來看,以MR配方於瓜類作物上 施用之安全性特質已有一較為具體之支持證據,其病害防治上之應用就永續農業之經 營而言確為一環境安全性良方。
The development of MR (methionine and riboflavin) formulation is a first successful attempt in the world of using photodynamic biocidal food constituents as main ingredients for the fungicide applications. One of the unique features of MR as a fungicidal formulation is that it is not likely to cause residual toxicity problems and thus is environmentally safe in the practical field application for disease control. The initial discovery of the microbial toxicity of MR was made by Tzeng and DeVay in 1984. During the past 10 years, a lot of efforts have been devoted to elucidate the involved molecular basis of the microbicidal activity; and a series of field trials have demonstrated the practical value of the developed concoction as a disease control agent. The formulation is currently on the process of commercialization in Taiwan, for the powdery mildew control on cucurbits, through the efforts of American Cyanamid Co. Taiwan Subsidiary However, so far the possible physiological or ecological impact of continued application of this novel fungicidal formulation remains largely to be explored. With this in mind, the purpose of this investigation was thus aimed at evaluating the possible physiological effects of a plant due to continued application of MR formulation. Using greenhouse grown muskmelons (Cucumis melon cv. Non-Fa No.38) as test meterial, possible important foliar function changes were monitored after the application of MR. Through the course of this study, none of the test plants showed observable morphological damages due to MR application. But, it was first noted that after each MR application, there was always a transient increase of ethylene production and proline contenet in treated foliar tissues indicating the presence of certain stressful effect due to the treatment. On the contrary, the stomatal conductivity as well as the contents of free amino acid, soluble protein, and chlorophyll were all not affected; and membrane permeability of the foliar tissues, as reflected by the electrolyte leakage detection, also seemed to remain normal. A followed SDS-PAGE analysis of the soluble proteins, however, revealed a transient increase of a 90kD protein in the MR treated foliar tissue. And a continued HPLC analysis of the free amino contents further indicated a transient surge of glycine content in accompany to the mentioned ethlene and proline increament. Although the plausible reason and the significance of changes of glycine metabolism all remained to be elucidated, the above results indicated clearly the lack of substantial, unrecoverable deteriorative effect on the vital function of test plants due to the MR application. As a photodynamic biocidal agent, the effectivity of MR formulation for disease control resides mainly upon the production of various activated oxygen species. The lack of detectable damaging effect from MR treated foliar tissues implicated the existence of efficient protective mechanisms for the toxigenic oxygen species in the plant tissue. In order to learn how the test plants survive the oxygen toxicity associated with MR application, the changes of enzymatic as well as nonenzymatic biomolecules known to be of great potential in contributing to the defense mechanism of the MR treated foliar tissues were examined. In Mr treated foliar tissue, we noted that the activities of ascorbate peroxidase, glutathione reductase, and superoxide dismutase were not affected throughout two consecutive MR applications. The activity of peroxidase activity, however, significantly increased. Isozyme analysis by isolelectric frcusing electrophoresis further indicated that the change was mainly due to the increased activity of an isozyme at pI 10.0. In contrast to this, a transient decrease of catalase activity was detected from the foliar tissues after each MR treatment. Activity of this enzyme generally resume the level as the control plants 3 days after MR application. Analysis of the contained isozymes by Native PAGE, however, indicated a change of electrophoretic mobility of these enzyme molecules. In the same experiment, results obtained from HPLC analysis further revealed the lack of effect of MR treatment on contents of reduced form glutathione and carotenoid in foliar tissues, although a slight but significant decrease of oxidized form glutathione and ascorbate were both detected from test leaves after MR application. From the above discussed evidences, it seems clear that certain changes of active oxygen defensing biomolecules do exist in MR treated foliar tissue. However, except peroxidase which appeared to have somewhat prolonged effect during a two consecutive weeks experimental periol, the effect on other tested enzymatic or non-enzymatic molecules all appeared to be instant and rather transient. The lack of prolonged effect among these test biomolcules suggested that the protective effect of test plants resided mainly on the preexisting oxygen toxicity defensing mechanisms. What worth to mention more is that the increased activity of peroxidase and an accompanied decrease of catalase activity appeared to be common feature recently known to be associated with certain plants with systemically acquired resistance (SAR). Although whether or not this might also apply to the case of MR treated plants awaited to be explored, the evidence provided by this investigation does support fully of the use MR as an environmentally safe fungicide for field application.
URI: http://hdl.handle.net/11455/31696
Appears in Collections:植物病理學系

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