應用Bacillus subtilis TKS1-1及SP4-17菌株防治草莓炭疽病

黃翔瑜

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應用Bacillus subtilis TKS1-1及SP4-17菌株防治草莓炭疽病

Application of Bacillus subtilis strains TKS1-1 and SP4-17 for the control of strawberry anthracnose

黃翔瑜 , Masters　　Advisor：黃姿碧　　

繁體中文

枯草桿菌；草莓；炭疽病；生物防治；細胞凋亡； Bacillus ； strawberry ； Colletotrichum gloeosporioides ； biocontrol ； apoptosis

Abstract │ Reference (125) │ Altmetrics

Abstract 〈TOP〉

由Colletotrichum gloeosporioides所造成的草莓炭疽病，嚴重影響草莓幼苗之生長，造成台灣及世界各地草莓產量上之損失，然而目前防治此病害之方法仍主要依賴化學藥劑，為了尋找對環境友善之防治策略，以微生物製劑防治此病害為一替代之選擇。已知Bacillus屬的細菌為常見之根圈有益微生物之一，其不僅可促進植株之生長，亦可產生抗生物質或誘發植物系統性抗性，抑制植物病害之發生。因此本研究旨在篩選台灣本土對於草莓炭疽病害具有防治潛力之Bacillus屬菌株。Bacillus subtilis菌株TKS1-1以及Bacillus megaterium 菌株SP4-17在本研究前期中證實對許多真菌性病原及Xanthomonas類細菌性病原具有拮抗之活性，且TKS1-1菌株可降低水稻白葉枯病及柑橘潰瘍病之發生，而本研究亦發現此兩株菌株對於草莓炭疽病菌亦較其他菌株具優良的抑制生長能力，故本研究以此兩株菌株進行後續試驗。前期研究中，結果顯示SP4-17菌株其屬種為B. megaterium，然近期Biolog系統之演進及資料庫之更新，故本研究中再以Biolog Generation III system、生理生化特性及核酸多型性圖譜分析重新進行SP4-17菌株之鑑定，試驗結果顯示SP4-17菌株與TKS1-1及B. subtilis標準菌株親緣關係較接近而與B. megaterium標準菌株關係較遠，故將SP4-17菌株屬種定為 B. subtilis。為瞭解TKS1-1及SP4-17菌株於草莓葉片上纏據之能力，研究中測定此兩菌株在葉片上每三日之族群動態，試驗結果發現此兩株菌株在施用後第21天其菌量仍可維持在105 CFU/ ml，與起初接種濃度相當。於溫室接種試驗結果中也證實，施用100倍稀釋之TKS1-1及SP4-17內生孢子發酵液可降低約50 %之罹病度。而B. subtilis TKS1-1以及SP4-17菌株之內生孢子濾液可抑制草莓炭疽病菌孢子發芽，且造成孢子畸形膨大。另可觀察到TKS1-1以及SP4-17培養濾液對草莓炭疽病菌之分生孢子具誘導染色質凝聚及細胞膜上磷脂絲胺酸外顯性現象等類似細胞程序性凋亡之指標性特徵，且亦發現其可抑制草莓炭疽病菌之能量代謝。除此之外，施用TKS1-1及SP4-17菌株內生孢子發酵液可促進草莓植株之生長。綜合上述結果，顯示拮抗微生物B. subtilis TKS1-1以及SP4-17具草莓炭疽病防治應用之潛力。

Parallel Abstract 〈TOP〉

Strawberry anthracnose caused by Colletotrichum gloeosporioides severely impacts the strawberry nurseries and greatly reduces yields and quality of strawberry in Taiwan and worldwide. The current commercial control measures for anthracnose disease rely mainly on chemical pesticides. Toward environmental friendly control strategies, application of microbial pesticides could be an alternative method. Bacillus species is representative genera of plant growth promoting rhizobacteria which not only promote plant growth but also act as biocontrol agents by producing antibiotics, triggering induced systemic resistance. The main objective of this study was to explore potential antagonistic Bacillus species native in Taiwan for the control of strawberry anthracnose. Bacillus subtilis strain TKS1-1 and strain SP4-17 previously were shown to antagonize against various pathogenic fungi and Xanthomonas species and strain TKS1-1 was reported to reduce disease incidence of citrus bacterial canker and rice bacterial blight. Here, we found that the two strains showed better antagonistic activity against anthracnose fungi of strawberry than other Bacillus species strains. Thus, they were selected for futher study. Previous data suggested the species attribute of strain SP4-17 was B. megaterium. Due to the renovation of the Biolog Microbial Identification system and the expanded Generation III database. The strain SP4-17 was subjected to identification by Biolog Generation III System, analysis physiological and biochemical characteristics and DNA polymorphism analysis by BOXA1R-derived gentic fingerprints. Results suggested that strain SP4-17 was close relative to the strain TKS1-1 and the type strain of B. subtilis, but different from the type strain of B. megaterium, thus was re-classified as B. subtilis.To investigate the colonization and survival ability of B. subtilis strains TKS1-1 and SP4-17 on leaves of strawberry plants, population dynamics of both strains were determined. Results indicated that at 21 days post-treatment of the two strains, 105 colony forming units per cm2 of strains TKS1-1 or SP4-17 were colonized on leaves of strawberry plants. The numbers of cells colonized were similar to that were inoculated. The disease severity of strawberry anthracnose was reduced up to 50 % when treatment with endospore formulations of B. subtilis TKS1-1 and SP4-17 diluted 100-fold. Application of culture filtrates from endospore formulations of B. subtilis TKS1-1 and SP4-17 inhibited spore germination, and caused aberrant spore and germ tube morphology of C. gloeosporioides. The culture filtrates were also found to trigger chromatin condensation and phosphatidylserine externalization, which were two typical features of the apoptotic-like program cell death and cause reduction in energy metabolism of C. gloeosporioides GH-A compared to the medium control, In addition, application of B.subtilis TKS1-1 and SP4-17 on strawberry plants promote growth of strawberry plants compared to the water and the medium controls. To summarize, B.subtilis TKS1-1 and SP4-17 are potential biocontrol agents for strawberry anthracnose.

Reference ( 125 ) 〈TOP〉

江迪蔚。2006。枯草桿菌Bacillus subtilis WG6-14 於檬果黑斑病防治之應用潛力與作用機制。國立中興大學植物病理系碩士論文。79 頁。
連結：
呂冠民。2011。柑橘潰瘍病菌生物膜與致病性及枯草桿菌群於其生物防治應用機制。國立中興大學植物病理系碩士論文。84頁。
連結：
林漢釗。2006。益生性枯草桿菌 Bacillus subtilis WG6-14在水稻栽培與病害管理上之應用性。國立中興大學植物病理系碩士論文。152頁。
連結：
林雅涵。2015。茶與百香果炭疽病之拮抗微生物鑑定及其於病害防治應用潛力。國立中興大學植物病理系碩士論文。90頁。
連結：
吳佳樺。2010。聚麩胺酸量產於枯草桿菌特殊功能生物殺菌劑發展之應用。國立中興大學植物病理系碩士論文。68頁。
連結：

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