The non-sterile propagation and mode of action of Streptomyces griseobrunneus S3 for controlling disease caused by Pythium aphanidermatum and Rhizoctonia solani AG4.

陸、中文摘要 Streptomyces griseobrunneus S3之非無菌增量培養及其於腐霉病菌與立枯絲核菌危害防治之作用機制 陳泰元 本研究之主要目的在於建立供Streptomyces griseobrunneus S3 (SGS3) 生物製劑應用培養液不需經高壓滅菌之非無菌增量培養系統 (NSPS) 及其於病害防治相關作用機制之探討。供試生物防治菌株SGS3為本研究室賴氏 (2003) 由柑橘根圈土壤所分離，過去之研究已證實其為極具發展潛力之生物防治菌株，同時具備優異之抗生活性、幾丁質與β-1,3-葡萄聚醣分解酵素活性以及產孢活性等，為其可發揮有效生物防治製劑功能之重要屬性。為發展其做為生物製劑的應用性，本研究室已建立一pilot級液態發酵量產技術平台，其所產製SGS3之孢子懸浮培養，產量可達5x1011 cfu/ml。本研究中非無菌增量培養系統 (NSPS) 之建立，主要目的在提供栽培業者一個簡易的自製 (DIY) 系統，俾能在使用前可將發酵槽所產製之孢子生質體 (biomass) 先行增量培養，除了將製劑中孢子加以活化，並可減少生物製劑施用所需之花費。本研究首先於無菌培養系統下進行天然培養基質初步篩選，供試穀物中以3% 粉頭做為主要營養源進行液態培養，SGS3產孢效果最好；而0.2% 尿素氮素源之添加，則對所產製培養液抗生活性表現之提升效果極為顯著。為建立非無菌增量培養技術，本研究進而利用未經高壓滅菌的培養基質進行系列的篩選測試，試驗中發現當以粉頭為主要培養基質時，於NSPS下SGS3之生長量顯因培養過程中有其他污染性微生物存在的影響，生長曲線有起伏波動之情形。就此問題，本研究繼而發現如在接種前將粉頭液體培養基先經煮沸處理5分鐘，則可有助降低培養液中其他微生物之干擾，並顯著提升SGS3生質體之產量。且在接種源應用、培養基緩衝能力以及培養基質組成等培養條件之最適化之後，此一污染性微生物存在所造成的干擾已不具顯著性，培養基質之煮沸處理似無必要性。在以粉頭為主要營養源配合100 mM K2HPO4與0.1% 蚵殼粉的最適化添加應用，可大幅提升SGS3的生長量，培養3天後菌量即可達2x1012 cfu/ml。其中K2HPO4添加對SGS3生長的促進作用，顯然與其所提供培養液的緩衝能力有關。此本研究所建立之最適化NSPS，除可適用於SGS3之外，對本研究室所保存之抗真菌性鏈黴菌菌株S1、S4與S31均有良好之增量培養效果，其孢子產量除S31菌株僅達約1010 cfu/ml外，S1與S4菌株之生長量均可達1012 cfu/ml水平。利用已建立的病害防治評估系統，本研究已證實上述NSPS所產製之增量培養液與利用發酵槽行無菌培養所獲得之發酵菌液比較，可有同等之病害防治效果，其做為生物防治製劑之應用性相當值得肯定。在對腐霉病菌與立枯絲核菌所造成危害之防治方面，本研究中證實以100倍稀釋之SGS3培養液於種植前2 (對腐霉病菌) 及4 (對立枯絲核菌) 天進行澆灌處理，相較於以水處理之對照組，可分別減少77% (對腐霉病菌) 與64% (對立枯絲核菌) 的發病率；且以種子被覆處理亦可得到同等優異的病害防治效果。另外值得注意的是，藉由種植前澆灌處理與種子被覆處理的配合應用，對此二病原之害，其相對於對照處理，發病率降低幅度均可達81% 以上，其防治效果與個別的推薦藥劑-滅達樂 (對腐霉病菌，施用濃度350 μg a.i./ml) 與賓克隆 (對立枯絲核菌，施用濃度250 μg a.i./ml) 已無顯著差異。本研究中SGS3病害防治效果之發揮與施用劑量明顯有關，其中SGS3孢子體之數量尤為關鍵，而培養液 (即培養濾液) 部分則雖其所含SGS3代謝產物的應用確實對兩供試病原真菌危害的抑制作用確有協力效果，然其於病害防治機制中應僅屬於次要之角色。供試SGS3當以人工接種方式處理腐霉病菌與立枯絲核菌之菌絲體後，於顯微鏡檢視下，已證實SGS3之孢子可迅速地發芽、生長並於病原菌菌絲體上纏據及侵入。此一超寄生作用已知可導致病原菌菌絲體之電解質滲漏迅速增加，且在處理後24小時之內，病原真菌菌絲體即有明顯之致死作用，隨幾丁質的添加已知可促進此一作用，而葡萄糖之添加則有抑制現象，此一結果顯示幾丁質與葡萄聚醣分解酵素之作用為遂行超寄生作用極具關鍵之特性。此外，由於此一致死作用在標的真菌菌絲體尚未出現明顯之崩解現象之前即已相當顯著，其中SGS3代謝物抗生性物質的參與仍不容忽略。唯不論如何，本研究中所見SGS3對標的病原真菌迅速且致死的超寄生活性，充分顯示SGS3如何幫助植物防範病原真菌的侵害。就病害防治效果而言，本研究進一步以番茄幼苗為模式系統，研究SGS3培養液澆灌處理對植物抗病性的影響。試驗結果證實番茄植株在經SGS3澆灌後，其葉片中即可測得PR-1 (病程相關蛋白-1) 基因的誘導表現，顯示植物抗病基因明顯有被誘導表現之情形。其中由比較澆灌SGS3培養液處理與澆灌培養濾液處理後，葉片中PR-1基因被誘導表現之差異情形，則再次顯示SGS3生質體存在的關鍵性。綜合上述，本研究之結果已可明白看出，包括超寄生作用、抗生活性與誘導植物抗病性等多重作用機制的發揮，為SGS3可表現優異之病害防治效果之主要原因，由研究中所提供之試驗結果，已證實其可達到與化學藥劑施用同等之病害防治效果。另外利用本研究所建立之NSPS除能成功地活化SGS3生質體，其於實際病害防治上的應用性也已獲得證實，確為產業應用上值得推薦之技術。柒、英文摘要 The non-sterile propagation and mode of action of Streptomyces griseobrunneus S3 for controlling diseases caused by Pythium aphanidermatum and Rhizoctonia solani AG4. Chen Tai-Yuan The main objectives of this investigation were to establish a non-sterile propagation system (NSPS) for biological control application of Streptomyces griseobrunneus S3 (SGS3) and to explore the mode of action of the disease control observed. The tested biological control agent SGS3, originally isolated by Lai (2003) from citrus rhizosphere, has been shown having great potential for biofungicide application. The main attributes known pertaining to its effectiveness as biocontrol agent included the excellence in chitin and β-1,3-glucan degradation, and as well the antibiotic and sporulating activities. And for the biofungicide application, a pilot scale liquid fermentation technique platform was established; the yield of spore suspension of SGS3 reached 5x1011 cfu/ml. The establishment of a NSPS discussed in this investigation was aimed to provide a grower do-it-yourself (DIY) system for the mass propagation and reactivation of the fermenter produced spore biomass right before application and thus to reduce the cost of the biofungicide application. In a preliminary screening trial where that sterile culture system was applied, the provision of wheat bran at 3% was found the best among the tested grain varieties as natural substrate for supporting the spore production of SGS3 in a broth culture system. Urea at 0.2% was found to be an appropriate nitrogen supplement for boosting up the antibiotic activity of the produced broth culture. For the serial trials conducted to establish the NSPS technique, the test bacterium was cultured with the test growth medium without autoclave sterilization. In a NSPS broth culture using wheat bran as the major growth substrate, the growth of SGS3 appeared to fluctuate during the culturing period mainly because the presence of certain microbial contaminants. A 5-minute boiling treatment of the wheat bran broth medium was found helpful in reducing the interference of the contaminating microbes thus provided significantly increased yield. However, with the optimization of the cultural substrate formulation, the applied inoculants, and the buffer capacity of the growth medium, the interference of the contaminating microbes became non-significant and the boiling treatment appeared not necessary. In an optimized cultural condition using wheat bran broth as major nutrient constituent, the growth of SGS3 was greatly improved by the addition of 100 mM K2HPO4 and 0.1% oyster shell powder; the spore yield of SGS3 reached approximately 2x1012 cfu/ml 3 days after incubation. The stimulatory effect of K2HPO4 addition appeared to be associated with the buffer capacity provided. In addition to SGS3, the optimized NSPS established appeared to work for Streptomyces sp. S1, Streptomyces sp. S4, Streptomyces saraceticus S31; the spore yield all reached 1012 cfu/ml level except that of S31 which yielded only to 1010 cfu/ml. The usefulness of the established system in Streptomyces sp. biofungicide application was clearly indicated further by the fact that the disease control effectiveness provided by the NSPS broth culture was equibalent to that by the original fermenter produced culture broth. For seedling damping offs caused by Pythium aphanidermatum and Rhizoctonia solani AG4, effective disease control was demonstrated. By drenching treatment with SGS3 broth cultures at 100X in dilution 2 days (for P. aphanidermatum) to 4 days (for R. solani AG4) before seeding, the percent infection of the two diseases was reduced by 77 to 64%, respectively, as compared to the water treated control. A comparable disease control effectiveness was also demonstrated when SGS3 was applied by seed coating. What worth to mention is that when the pre-seeding drenching treatment was combined with seed coating, the percent infection of both targeted diseases was reduced over 81%; the protective effect was equivalent to that by the compared chemical treatment- metalaxyl (applied at 350 g a.i./ml for P. aphanidermatum) and pencycuron (applied at 250 g a.i./ml for R. solani AG4). The effectiveness of disease control appeared to be dosage dependent which relies primarily on the presence of SGS3 spore propagules. The role of metabolites present in the broth culture (i.e. cultural filtrate) appeared to be minor as regards to the disease control demonstrated although its presence did contribute some additive effect in counteracting the fungal invasion. Upon artificial inoculation of SGS3, the microscopic examination revealed that the applied bacterial spores germinate readily, grew on, coiled up and even penetrated the mycelia of P. aphanidermatum and R. solani AG4. The mycoparasitism led to rapid increase of electrolytes from the host fungal mycelia, and the parasitized mycelia appeared to be killed within 24 hours. The mycoparasitic effect was manifested by the formation of eroded concave lesion reflecting the functioning of chitin/glucan degrading enzymes; the activity was greatly stimulated by the addition of chitin whereas was repressed by the addition of glucose. The killing of target fungi, as revealed by fluorescen diacetate/propidium iodide vital staining, was detected before dismantling of the fungal cell became apparent indicating the involvement of antibiotics during the process. The fast and lethal mycoparasitic activity well illustrated how SGS3 helps plants fight with the fungal pathogens. As for the disease control, the effect of SGS3 application on the disease resistance was investigated using tomato as a model. An enhanced expression of PR-1 (pathogenesis related protein 1) gene, indicating the induction of resistance gene expression, was detected consistently from foliar tissue after drenching treatment of SGS3. As the induced PR-1 gene expression was detected from the culture broth treated rather than the culture filtrate treated plants, the existence of SGS3 propagules again appeared to be critical. The data herein presented indicates clearly the disease control by SGS3 is a result of multiple mode of action including mycoparasitism, antibiotic activity, and induced resistance. The effective disease control equivalent to that by chemical fungicides was demonstrated. The success of NSPS development warrant the innundative application of the viable, active SGS3 propagules in practical application.目錄 壹、前言--------------------------------------------------------------------------------------------------------1 貳、前人研究--------------------------------------------------------------------------------------------------4 參、材料與方法----------------------------------------------------------------------------------------------11 一、供試藥品試劑來源----------------------------------------------------------------------------------11 二、培養基種類-------------------------------------------------------------------------------------------11 (一) 幾丁質培養基 (chitin medium) 之製備----------------------------------------------- 11 (二) 馬鈴薯蔗糖瓊脂培養基 (potato sucrose agar, PSA) 之製備--------------------11 (三) 水瓊脂培養基 (water agar, WA) 之製備--------------------------------------------- 12 三、供試植株、菌株來源與培養----------------------------------------------------------------------12 四、SGS3孢子懸浮液接種源製備-------------------------------------------------------------------13 五、培養濾液抗生活性檢測----------------------------------------------------------------------------13 六、無菌培養下培養條件初步篩選-------------------------------------------------------------------13 (一) 不同穀物基質對菌量增殖之影響--------------------------------------------------------13 (二) 培養液pH值對菌量增殖之影響---------------------------------------------------------14 (三) 不同濃度尿素的添加對抗生活性表現之影響-----------------------------------------14 七、非無菌增量培養系統 (NSPS) 之建立-培養條件之最適化測試-------------------------14 (一) 不同濃度粉頭培養液對菌量增殖之影響-----------------------------------------------14 (二) 不同接種濃度對菌量增殖之影響--------------------------------------------------------14 (三) chitosan之添加對菌量增殖之影響------------------------------------------------------15 (四) 磷酸氫二鉀之添加對菌量增殖、抗生活性表現與儲存安定性之影響------------15 (五) 培養液事前煮沸處理對菌量增殖、培養液酸鹼值變化及抗生活性表現之影響------------------------------------------------------------------------------------------------- 15 (六) 蚵殼粉之添加對菌量增殖、培養液酸鹼值變化與儲存安定性之影響------------16 (七) 不同菌齡接種源對菌量增殖之影響-----------------------------------------------------16 (八) 不同鏈黴菌菌株於NSPS最適化培養條件下之培養效果--------------------------16 八、SGS3於甘藍幼苗猝倒病及立枯病防治之應用性-------------------------------------------17 (一) 以不同穀物培養基所產製之SGS3培養液澆灌處理對甘藍幼苗生長之影響--17 (二) 種植前不同濃度SGS3澆灌處理對甘藍幼苗猝倒病與立枯病之防治效果-----17 (三) SGS3培養菌液、NSPS增量培養液、菌體及培養過濾液對甘藍幼苗猝倒病與立枯病之防治效果比較------------------------------------------------------------------- 18 (四) 種子被覆與澆灌處理對甘藍幼苗猝倒病與立枯病之防治效果--------------------18 九、SGS3於病害防治之相關作用機制------------------------------------------------------------19 (一) 以SGS3培養菌液、培養濾液與孢子懸浮液之添加處理對P. aphanidermatum與R. solani AG4菌絲體細胞膜通透性之影響---------------------------------------19 (二) 幾丁質與葡萄糖之添加對SGS3所造成P. aphanidermatum與R. solani AG4菌絲體細胞膜滲漏作用之影響---------------------------------------------------------- 20 (三) 幾丁質之添加前處理時間對SGS3所造成P. aphanidermatum與R. solani AG4菌絲體細胞膜滲漏作用之影響-----------------------------------------------------20 (四) SGS3之超寄生作用對P. aphanidermatum與R. solani AG4菌絲體生長與發育之影響--------------------------------------------------------------------------------------20 (五) SGS3對P. aphanidermatum與R. solani AG4超寄生作用之顯微鏡檢視----21 (六) SGS3對P. aphanidermatum及R. solani AG4菌絲體細胞活性之影響-------21 (七) 以SGS3培養菌液澆灌處理對植物系統性抗病性之誘導作用--------------------22 1. 供試植物與SGS3澆灌處理-----------------------------------------------------------22 2. 供試植物RNA的抽取-------------------------------------------------------------------22 3. RNA電泳分析-----------------------------------------------------------------------------23 4. 蕃茄pathogenesis related protein-1 (PR-1) 及phenylalanine ammonia- lyase (PAL) 探針之製------------------------------------------------------------------ 23 5. 北方雜合作用-----------------------------------------------------------------------------24 肆、結果----------------------------------------------------------------------------------------------------26 ㄧ、無菌培養下培養條件初步篩選----------------------------------------------------------------- 26 (一) 不同穀物基質對菌量增殖之影響--------------------------------------------------------26 (二) 培養液pH值對菌量增殖之影響---------------------------------------------------------26 (三) 不同濃度尿素的添加對抗生活性表現之影響-----------------------------------------26 二、非無菌增量培養系統 (NSPS) 之建立-培養條件之最適化測試------------------------- 27 (一) 不同濃度粉頭培養液對菌量增殖之影響-----------------------------------------------27 (二) 不同接種濃度對菌量增殖之影響--------------------------------------------------------27 (三) chitosan之添加對菌量增殖之影響------------------------------------------------------27 (四) 磷酸氫二鉀之添加對菌量增殖、抗生活性表現與儲存安定性之影響------------28 (五) 培養液事前煮沸處理對菌量增殖、培養液酸鹼值變化及抗生活性表現之影響------------------------------------------------------------------------------------------------- 29 (六) 蚵殼粉之添加對菌量增殖、培養液酸鹼值變化與儲存安定性之影響------------30 (七) 不同菌齡接種源對菌量增殖之影響-----------------------------------------------------31 (八) 不同鏈黴菌菌株於NSPS最適化培養條件下之培養效果--------------------------31 三、SGS3於甘藍幼苗猝倒病及立枯病防治之應用性-------------------------------------------31 (一) 以不同穀物培養基所產製之SGS3培養液澆灌處理對甘藍幼苗生長之影響--31 (二) 種植前不同濃度SGS3澆灌處理對甘藍幼苗猝倒病與立枯病之防治效果-----32 (三) SGS3培養菌液、NSPS增量培養液、菌體及培養過濾液對甘藍幼苗猝倒病與立枯病之防治效果比較------------------------------------------------------------------- 32 (四) 種子被覆與澆灌處理對甘藍幼苗猝倒病與立枯病之防治效果--------------------33 四、SGS3於病害防治之相關作用機制-------------------------------------------------------------34 (一) 以SGS3培養菌液、培養濾液與孢子懸浮液之添加處理對P. aphanidermatum與R. solani AG4菌絲體細胞膜通透性之影響---------------------------------------34 (二) 幾丁質與葡萄糖之添加對SGS3所造成P. aphanidermatum與R. solani AG4菌絲體細胞膜滲漏作用之影響---------------------------------------------------------- 34 (三) 幾丁質之添加前處理時間對SGS3所造成P. aphanidermatum與R. solani AG4菌絲體細胞膜滲漏作用之影響-----------------------------------------------------35 (四) SGS3之超寄生作用對P. aphanidermatum與R. solani AG4菌絲體生長與發育之影響-------------------------------------------------------------------------------------- 36 (五) SGS3對P. aphanidermatum與R. solani AG4超寄生作用之顯微鏡檢視-----36 (六) SGS3對P. aphanidermatum及R. solani AG4菌絲體細胞活性之影響--------37 (七) 以SGS3培養菌液澆灌處理對植物系統性抗病性之誘導作用--------------------37 1. 對蕃茄植株PR-1與PAL基因表現之誘導作用-----------------------------------37 2. 對蕃茄幼苗PR-1及PAL基因表現之誘導作用-----------------------------------38 伍、討論-------------------------------------------------------------------------------------------------------39 陸、中文摘要-------------------------------------------------------------------------------------------------49 柒、英文摘要-------------------------------------------------------------------------------------------------52 捌、引用文獻-------------------------------------------------------------------------------------------------56 玖、圖表說明-------------------------------------------------------------------------------------------------6