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標題: 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
Identification of antagonistic microorganisms against anthracnose on tea and passion fruit and their potential application in disease control
作者: Yia-Han Lin
關鍵字: 炭疽病;枯草桿菌;生物防治;anthracnose;Bacillus;biocontrol
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茶葉與百香果在國際上為重要大眾飲用作物之一,由Colletotrichum spp.所引起的茶炭疽病 (brown blight of tea) 以及百香果炭疽病 (anthracnose of passion fruit) 是茶樹與百香果上重要真菌病害之一,危害嚴重時會造成產量損失。目前防治方法是以施用化學藥劑為主,然而不當使用化學藥劑的結果會造成許多抗藥性菌株的出現以及對人類和環境造成負面的影響,所以生物防治是一個良好的替代性防治方法,而Bacillus 屬以及 Streptomyces屬之菌株已知為常見促進植物生長之根圈有益微生物,不僅可以促進植物生長,甚至具有抗生活性及誘導系統性防禦反應等生物防治潛力。因此我的研究目的為篩選鑑定來自田間之有益微生物,並探討其防治茶及百香果炭疽病以及促進茶及百香果幼苗生長之應用潛力,並探討其防治之機制。自南投、高雄及台中根圈土壤中篩選分離出之菌株Bacillus sp. AM4-1、CA-1、151B1以及151B4由生理生化測試、Biolog System III、16S rRNA及核酸多型性圖譜分析鑑定均為Bacillus subtilis;Streptomyces sp. UN3S2、PES4經由16S rRNA只能初步鑑定為Streptomyces sp.,上述菌株與生理與分子病理研究室提供之菌株B. subtilis TKS1-1和S. griseobrunneus S3共八株菌株,對茶炭疽病原菌C. gloeosporioides TE04菌絲生長及孢子發芽有較優異的抑制效果。且施用B. subtilis 151B1培養液於百香果扦插苗上,在不論接種炭疽病菌與否之條件下均可提升其存活率。此外,處理B. subtilis TKS1-1及B. subtilis 151B1培養濾液於百香果炭疽病菌C. karstii N-PF1孢子懸浮液中,會造成其細胞凋亡且染色質無法凝聚之現象,亦會降低炭疽病原真菌粒線體膜潛勢以及呼吸作用能量代謝之現象,推測其培養濾液可能與誘導造成細胞程序性死亡有關。此外C. karstii N-PF1孢子懸浮液處理B. subtilis TKS1-1及B. subtilis 151B1培養濾液12及24小時之後,發現C. karstii N-PF1菌絲有畸形生長或孢子未發芽之現象。在B. subtilis TKS1-1, B. subtilis151B1, S. griseobrunneus S3和Streptomyces sp. PES4四株菌株中,以B. subtilis 151B1生長促進能力最佳,施用其培養液於青心烏龍扦插苗生長上處理八周後,相較其他處理組其葉長及葉厚可增加約2倍,在百香果幼苗上可增加新葉數約2.6倍、節數增加約2倍、葉寬約2倍及在植株莖鮮種約1.5倍。Streptomyces sp. PES4 則是施用其培養液於青心烏龍扦插苗生長上處理八周後,相較其他處理組其株高增加約2倍。總結上述試驗結果,拮抗微生物B. subtilis 151B1對於炭疽病菌具有優異的拮抗效果,且施用其培養液於百香果扦插苗上,在不論接種炭疽病菌與否之條件下均可提升其存活率,且對茶及百香果幼苗具有生長促進之現象,推測B. subtilis 151B1為具有生物防治潛力之菌株,此外我們結果亦發現B. subtilis TKS1-1及B. subtilis151B1之培養濾液可能會誘導炭疽病菌細胞程序性死亡,導致其菌絲畸形生長及抑制孢子發芽之能力。

Tea (Camellia sinensis) and passion fruit (Passiflora edulis) are the most consumed beverages in the world. Brown blight of tea and anthracnose of passion fruit caused by Colletotrichum gloeosporioides are both devastating diseases hindering their production. Extensive use of fungicides has led to serious development of resistance in pathogen populations and caused negative consequences for human health and the environment. Biological control has been taken as an alternative to disease control by synthetic pesticide. Bacillus and Streptomyces species are representative genera of plant growth promoting rhizobacteria which not only promote plant growth but could also act as biocontrol agents by producing antibiotics, triggering induced systemic resistance. The main objectives of my study are to select and identify native antagonistic microorganisms against pathogens of brown blight of tea and anthracnose of passion fruit, and to investigate their potential application in disease control and growth promotion and to investigate the putative control mechanisms. Among seventy isolates which were isolated from rhizospere soils and collected from Nantou county, Kaohsiung city and Taichung city in this study, strains AM4-1, CA-1, 151B1 and 151B4 were classified as Bacillus subtilis group based on the analysis of 16S rRNA sequences, DNA polymorphism, physiology and biochemistry tests and the analysis by Biolog System III. Strains UN3S2 and PES4 were classified as Streptomyces species based on the analysis of 16S rRNA sequences. Eight strains including B. subtilis TKS1-1, Streptomyces griseobrunneus S3 and strains B. subtilis AM4-1, CA-1, 151B1, 151B4 and Streptomyces sp. strain UN3S2 and PES4 showed antagonistic effect on mycelial growth and conidial germination of C. gloeosporioides TE04. Application of B. subtilis 151B1 culture broth increased survival rates of passion fruit cuttings with or without the challenging of anthracnose fungi. Culture filtrates from strains 151B1 and TKS1-1 resulted in cell death and chromatin fragmentation of C. karstii N-PF1. The treatment of culture filtrates from strains 151B1 and TKS1-1 were also found to cause reduction in mitochondrial membrane potential and energy metabolism of Colletotrichum karstii N-PF1 compared to the medium control, suggesting its function in triggering apoptotic-like cell death. In addition, the aberrant hyphal morphology, non germinated conidia were observed for C. karstii N-PF1 on leaves of passion fruit while 12hrs and 24hrs post-challenging with the culture filtrates. Among four strains, TKS1-1, 151B1, S3 and PES4, B. subtilis 151B1 was found to show the superior enhance the leaf length and thickness (for 2-fold increase) of Chin-Shin Oolong seedlings 8 weeks post-treatment, and in numbers of leaves (2.6-fold increase), nodes (2-fold increase), leaf width (2-fold increase), and shoot fresh weight (1.5-fold increase) of passion fruit seedlings. In addition, Streptomyces sp. PES4 showed the greatest effect on promotion of plant height (2-fold increase) of Chin-Shin Oolong seedlings compared to the other treatments. In conclusion, B. subtilis strain 151B1 showed superior antagonistic activity against anthracnose fungi and enhancing the survival rates of passion fruit cuttings and with or without challenging with anthracnose fungi, and exhibited the greatest growth promotion of seedlings of tea and passion fruit. Suggesting it's potential as a biocontrol agent. Our findings also suggest that culture filtrates of B. subtilis TKS1-1 and 151B1 caused aberrant hyphal morphology and inhibit germination of anthracnose fungi which may part attribute to their ability in triggering program cell death of fungi.
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