Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89357
標題: 鏈黴菌Streptomyces sp. S1菌株作為木瓜疫病Phytophthora palmivora防治生物製劑之應用潛力
Potential of using Streptomyces sp. strain S1 as a bioagent for the control of papaya root rot disease caused by Phytophthora palmivora
作者: Chon-Leong Chan
陳俊良
關鍵字: 鏈黴菌
木瓜疫病
生物防治
加成作用
殺菌劑
Streptomyces
papaya root rot disease
bioagent
additive effect
fungicides
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摘要: 供試鏈黴菌Streptomyces sp. strain S1菌株為賴氏 (2003) 由市售栽培用泥碳土材料分離所得,已證實其對部分土壤傳播性真菌及類真菌等病原具優異的防治潛力,其病害防治應用效果亦證實與其幾丁質分解酵素及β-1,3-葡萄聚醣分解酵素相關,使其具有超寄生及產生二次代謝物之特性。本研究目的係嘗試將具有多種抑制病原機制之鏈黴菌與殺菌劑混合應用對卵菌病害之防治潛力,以降低卵菌綱病原抗藥性的產生及減低化學藥劑使用量。本研究中以對峙培養生物活性測試,證實S1對番椒疫病菌 (Phytophthora capsici)、木瓜疫病菌(Phytophthora palmivora),甘藍幼苗猝倒病菌 (Pythium aphanidermatum) 及草莓炭疽病菌 (Colletotrichum gloeosporioides) 顯現優異的抑制效果,且其抑制性與S1的接種時機有關。於馬鈴薯蔗糖瓊脂培養基 (potato sucrose agar, PSA) 測試供試鏈黴菌S1對27種供試藥劑的耐受程度,顯示除了賽普護汰寧、依普同、蓋普丹、四氯異苯腈及腈硫醌外,對其餘的22種供試藥劑皆具有一定程度的耐受性,耐受濃度甚至高達1000 ppm。進一步在生體外測試S1與七種供試藥劑混合處理對P. palmivora的孢囊及游走子發芽之抑制效果,就孢囊發芽之抑制性而言,滅達樂、座賽胺、安美速與依得利四種供試殺菌劑單獨處理效果均不佳;另外氟比來、亞托敏及達滅芬三種供試殺菌劑,對孢囊發芽均表現不等程度相當優異隨劑量而提昇的抑制效果。七種供試藥劑與S1混合處理對孢囊發芽的抑制效果均看不出有加成效果,然而提前10小時先單獨處理上述七種供試藥劑之孢囊發芽抑制效果均顯著提升,且在S1添加混合處理對七種供試殺菌劑藥效加成效果均極為顯著;在對游走子發芽的抑制效果方面,S1添加處理下,七種供試藥劑的藥效則均見明顯提高。接著檢視四種供試藥劑滅達樂、座賽胺、氟比來及達滅芬不同濃度對P. palmivora孢囊及游走子發芽管菌絲生長之影響,四種供試藥劑單用均可表現相當程度之抑制效果,抑制效果也均有隨著處理濃度增加而有不等程度提高的現象,當S1與供試藥劑混合應用時,10 ppm之滅達樂、氟比來及10、30、100 ppm之達滅芬的發芽管菌絲生長抑制率可見有明顯提昇之效果。供試日陞品種木瓜植株經以稀釋100、200、500倍之S1培養液每週兩次澆灌處理,連續澆灌5次後植株地上部平均高度與水澆灌處理之對照組明顯提高2.6至5.5 cm,此外一併檢視供試植株的根部生長鮮重,量測結果顯示處理S1培養液的根部鮮重顯著提高8.6至14.1 g,各處理間以S1培養液稀釋100倍澆灌生長促進效果最為顯著。後續測試S1培養液澆灌處理對木瓜幼苗疫病菌感染之防治效果,在接種病原菌前48小時先以稀釋100倍S1培養液行澆灌處理之木瓜幼苗,其存活率為所有處理中最高,與對照組相較顯著提高了約61.3%。再者,100 ppm濃度之滅達樂、氟比來、安美速及達滅芬四種供試殺菌劑,分別混合添加或不添加S1培養液行澆灌處理,試驗結果顯示,以S1培養液單用行澆灌處理,其存活率約為46.7%,相較之下以水澆灌的對照組只有26.7%存活。S1培養液與滅達樂或氟比來處理組其木瓜存活率為100%;而安美速與達滅芬處理組,其木瓜存活率則分別提高至96.7及86.7%,防治效果提高仍相當顯著。將四種供試藥劑濃度下調至10 ppm,以滅達樂防治效果最佳,幼苗存活率為97.3%,其他三種藥劑防治效果以幼苗存活率為指標則依次為氟比來80.7%、安美速61.3%、及達滅芬41.7%,與S1培養液混合添加處理下,四種供試藥劑之應用效果,其木瓜幼苗存活率皆為90%以上。同樣以溫室種植日陞種木瓜幼苗為測試材料,S1培養液與殺菌劑澆灌處理時機對防治效果之影響,試驗結果顯示S1培養液的添加施用,不論在接種前處理,或是接種後處理,滅達樂與達滅芬兩藥劑的防治效果皆獲得大幅提升。繼而以電導度測定儀檢測S1培養液之添加處理對P. palmivora菌絲體細胞膜通透性之影響,試驗結果證實於接種後2-5天之間維持相當恆定快速上升之趨勢。接種S1培養液處理24小時後生長出之菌絲並已經完成其在P. palmivora菌絲體及孢囊上之纏據入侵作用。另參考Cox試驗流程,以AlamarBlue®染劑做為分子探針,檢測S1培養濾液添加處理對P. palmivora能量代謝之影響,螢光反應結果證實,其對還原力之降低效果與市售呼吸作用抑制性殺菌劑亞托敏及賽座滅之效果相近。綜合上述,本研究中顯示鏈黴菌S1對木瓜幼苗疫病P. palmivora極具防治潛力,其作用機制可能包括超寄生作用及抗生活性等,除此之外,證實S1與低濃度卵菌用殺菌劑混合處理具有優異的防治效果,配合病害綜合防治管理體系,藉此降低農業栽培中的用藥成本、減少藥劑對環境的污染及破壞。
The antagonistic Streptomyces sp. strain S1 isolated from commercial peat moss (Lai, 2003) was shown of great potential as a microbial bioagent for the control of plant diseases – especially those caused by soil borne fungal/ fungal-like pathogens. The effectiveness of disease control was known to due greatly in part to the mycoparasitic effect – an unique characteristic of the bacteria associated with its excellence in chitinase and glucanase activites. The development of a bioagent for the disease control is badly needed because the great concern on the pesticide reduces, which appeared to be of particular importance because of the widespread existence of resistance of oomycete pathogens against various fungicides recommended for disease control. The main objective of this study was to explore the feasibility of integration of biological and/or chemical control measurement for the practical application for the disease control. The primed goal of using such an integrated strategy is to assure the effectiveness of disease control use. Additional benefit includes reduction of chemical residues, environmental impact and risk of resistance. A synergistic effect of the disease control by the integration of tested biofungicide is anticipated. By dual culture bioassay, the S1 broth culture were shown to be inhibitory on the mycelial growth of Phytophthora capsici, Phytophthora palmivora, Pythium aphanidermatum and Colletotrichum gloeosporioides. The observed inhibitory effect appeared to be dose dependent. A preliminary trial of tolerance of Streptomyces sp. strain S1 against fungicides was performed. Among 27 fungicides representative to different mode of action classified by Fungicide Resistance Action Committee (FRAC) tested, the tested Streptomyces strain S1 was able to grow on Potato sucrose agar (PSA) amended with tested fungicides except that for cyprodinil+fludioxonil, iprodione, captan, chlorothalonil and dithianon. In vitro, augmentation of S1 broth culture supplementation on the inhibitory effectiveness of selected oomycete fungicides on sporangial and zoospore germination of P. palmivora, inhibitory effectiveness of the tested fungicides metalaxyl, zoxamide, amisulbrom and etridiazole were poor. Inhibitory effectiveness of the tested fungicides fluopicolide, azoxystrobin and dimethomorph were good when concentration increased. The mixed application of fungicides together with S1 showed no additive effect on the germination of sporangia of P. palmivora. Also co-application of Streptomyces sp. strain S1 and fungicides 10 hours prior to inoculation of P. palmivora shows effective inhibition on germination of sporangia. For the inhibitory effect of germination of zoospores, 7 fungicides were significantly increased when treatment with S1. For P. palmivora, the inhibitory effect on sporangia and zoospore germ tube development, the inhibitory effect metalaxyl, zoxamide, fluopicolide and dimethomorph were improved when concentration increased. The mixed application 10 ppm of metalaxyl, fluopicolide or 10, 30 and 100 ppm of dimethomorph together with S1 showed additive effect on the development of germ tube inhibitory effect on mycelial growth was observed. Tested papaya (Carica papaya cv. Sunrise) seedlings were drenching treated twice per week each with S1 broth culture at 100, 200 and 500 of dilution for 3 consecutive weeks, the height of seedlings were significantly increased about 2.6 to 5.5 cm, in addition, the fresh weight of seedlings root were significantly increased about 8.6 to 14.1. Promoting effect of 100-dilution of S1 broth culture was the best in all of the treatment. The control of P. palmivora infection on papaya seedlings by Streptomyces sp. strain S1. The S1 broth culture at the 100-dilution was applied by drenching 48 hours before pathogen inoculation, the survival of seedlings was significantly increased about 61.3% compared with water treated control. The control of P. palmivora infection on papaya seedlings by tested 100 ppm of fungicides with or without the supplementation of S1. The survival of seedlings of S1 treatment about 46.7%, whereas that of the compared water treated control was only 26.7%. The survival of seedlings of metalaxyl or fluopicolide treated with S1 were 100%, the compared amisulbrom and dimethomorph treated with S1 were 96.7% and 86.7% respectively. The tested concentration of fungicides decreased to 10 ppm, survival of seedlings was about 97.3% by metalaxyl treatment without S1, the compared fluopicolide, amisulbrom and dimethmorph were 80.7%, 61.3% and 41.7% respectively. The survival of seedlings significantly increased to 90% were treated fungicides with S1. Then, the effect treatment timing on the effectiveness of P. palmivora infection control on papaya seedlings by fungicide/S1 biofungicide combined application. Whether before or after inoculation of P. palmivora, the survival of seedlings of treatment with S1 was better than water treated control. Upon S1 broth culture, an increased electrolyte leakage was detected from the test samples 2 to 5 days after treatment. Inoculation of S1 for 24 hours, that the applied bacterial spores grew on, coiled up and penetrated the mycelia of P. palmivora. Furthermore, with the addition of AlamarBlue® as indicative molecular probe, effect of the culture filtrate from S1 on the energy metabolism of P. palmivora comparable to that by azoxystrobin and cyazofamid treatment was demonstrated. Such combined use of biocontrol agent with fungicides can reduce the dosage of fungicides, thereby reducing environmental risks. The potential of using strain S1 as an aid for disease control, appears to be possible mechanisms, involved worth further attention.
URI: http://hdl.handle.net/11455/89357
文章公開時間: 10000-01-01
Appears in Collections:植物病理學系

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