Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/30815
標題: 放線菌Streptomyces saraceticus No. 31菌株所產生抗生物質理化與生 物特性之研究
Studies on the antibiotic production by the No.31 isolate of Streptomyces saraceticus, and its biological and biochemical properties
作者: 陳欣孝
Chen, Hsin-Hsiao
關鍵字: Streptomyces saraceticus;放線菌;antibiotic;抗生物質
出版社: 植物病理學系
摘要: 
本研究之主要目的在探討 Streptomyces saraceticus SS 31 菌株所產
生抗生物質之理化與生物特性、對病原菌之作用機制、及其在生物防治上
之實用性。 供試菌株 SS 31 為分離自幾丁質自然添加物之本土性拮抗菌
,其可分泌幾丁質分解酵素並對南方根瘤線蟲具優異之防治效果。利用種
子誘釣法評估其對帶菌土中立枯絲核菌之拮抗能力,證實在病害防治上具
應用潛力,進而以對峙培養發現本菌對許多植物病原性真菌及細菌具有廣
泛性之拮抗作用,特別是卵菌綱的 Pythium。利用 Pythium
aphanidermatum 為測試病菌,於 PSA平板上測試菌株抗生活性,發現在
pH 7.0 時、 30 ℃之培養條件下抗生活性最好,無論以 PSB 或
Czapek''medium 行液體培養時, 充足的通氣量有利於抗生物質的產生。
以Czapek''s medium 為基礎培養基,發現含氨基鹽( ammonium salts )
的氮素源對於抗生物質之產生較為有利。九種供測試之碳素源中,則以葡
萄糖、甘露糖、果糖、半乳糖、甘油與甘露糖醇為優, 另培養基中碳、
氮比例的改變及添加 Zn、 Fe、Mn、Cu、Ca、NaI、H3BO3、Na2MoO4 等礦
物元素對生長與抗生活性均無顯著影響。使用 9 種穀類研磨成粉做為培
養基質行液體培養,發現糙米、燕麥、高粱、玉米及薏仁等材料,在液體
培養過程中皆可測得抗生物質的產生,其中玉米及高粱最高。將放線菌所
產生抗生物質經 Waters 公司生產之 Sep-Pak C18 column 吸附及濃縮後
,利用高效液相層析儀( HPLC )層析分離, 並以光電二極體( PDA )
檢測器在 242 nm 波長下檢測分離結果,分離液經回收測定其拮抗活性,
結果發現在第 11 分鐘出現 A242 吸收高峰之樣品具有強拮抗活性,此一
吸收峰應用 Millennium 2010 Chromatography Manager (Waters
) 資料系統中之Spectral ContrastTM 技術分析,證實此吸收峰具有高
純度,回收此一分離樣品,以波長277 nm 之光線激發,可檢測得一波長
335 nm 螢光放射, 此抗生物質的分子量判定應小於 3 KDa,並在 pH
3.0~ 9.0 及高溫 100 ℃下 20 分鐘,仍具拮抗活性。探討抗生物質之作
用機制, 發現測試大部分病原真菌之菌絲生長與孢子發芽均受抑制,
以顯微鏡檢Pythium 之菌絲形態發現有生長不正常彎曲之現象,就炭疽病
菌而言,孢子懸浮液經抗生物質處理後會抑制其分生孢子發芽,並對其細
胞膜之通透性與細胞壁結構造成傷害。於溫室試驗中對 SS 31 之生物防
治應用性加以評估,發現施用 SS 31 孢子懸浮液於栽培介質中, 對甘藍
幼苗猝倒病菌 P. aphanidermatum 及立枯絲核菌 R. solani AG-4
(Cabbage-isolate ) 頗具防治成效,不只提高種子萌發率及減少幼苗死
亡率,並且對植株生長有明顯之促進作用,包括幼苗株高、根長及乾重等
均有增加之現象,在植物病害的生物防治上為極具發展潛力的微生物。

The investigation explored the physiological and biological
characteristicsassociated with the antibiotic production by
Streptomyces saraceticus isolate31, the possible mode of action
of the antagonism against fungal pathogen, andits potential
application as biopesticide for controlling plant diseases.
Thetested antagonistic bacterial isolate was known to be a
chitin degrader andhas been used for the control of Meloidogyne
incognita. By seed baiting anddual culture assay, it was
found to be effective in reducing the inoculumpotential of
certain AG group of Rhizoctonia solani and antagonistic
againstfairly wide spectrum of phytopathogenic fungi and
bacteria. The antagonisityagainst members of Oomycetes
including various Pythium species was especiallyworth noting.
Using Pythium aphanidermatum as a test target, antibiosis
oftest bacteria grown on PSA was found better expressed at pH
7.0 and at 30 ℃.The provision of adquate aeration greatly
favored the production of theantibiotics in either PSB or
Czapek''s broth. Using Czapek''s broth as basalmedium, ammonium
salts appeared to be superior source of nitrogen then nitratefor
supporting the antibiotic production. Among 9 tested carbon
sources,glucose, mannose, fructose, galactose, glycerol and
sorbitol appeared to besuitable for supporting the bacterial
growth and antibiotic production. Thegrowth and antibiotic
production were not affected by change of C/N ratio orthe
supplementation of various mineral elements including Zn, Fe,
Mn, Cu, Ca,NaI, H3BO3 and Na2MoO4. Powderized rice, oat,
sorghum, corn and coxi,especially corn and sorghum, were
found among 9 tested natural grains thebetter ones for
supporting growth and antibiotic production. The
antibioticproduced in broth culture containing sorghum powder
as culture substrate wasextracted by Sep-Pak (C18) separation
system (Waters) and further resolved andpurified by high
performance liquid chromatography (HPLC). The
antibioticfraction appeared to have an absorption peak at
242 nm as shown by a PDAmonitor. Spectrofluorometry of the
purified sample further indicated itsability to emit 335 nm
fluorescence when a 277 nm excitation illumination wasprovided.
The antibiotic appeared to be less than 3 KDa in molecular
weight.It was stable at pH ranged from 3 to 9 and at 100 ℃ for
20 min. When appliedto a potential target fungus, its mycelial
extension was arrested and sporegermination stopped.
Microscopic observation revealed that, for
Pythiumaphanidermatum, abnormal curving were quite common.
Increased electrolyteleakage was detected in conidial
suspension of Colletotrichum gloeosporioidesafter the
antibiotic treatment. The damages on membrane function and cell
wallformation were both implicated. The soil application of
spore preparation ofthe antagonist was found to be effective in
controlling infection of cabbageseedlings by P. aphanidermatum
and R. solani (AG-4, cabbage isolate) in thegreenhouse. It
increased not only the number of survival stands, but also
thegrowth vigor of the seedlings. The potential application of
tested isolate asbiopesticide for disease control deserved more
attention.
URI: http://hdl.handle.net/11455/30815
Appears in Collections:植物病理學系

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