應用Bacillus amyloliquefaciens PMB01 防治甜椒細菌性斑點病

Application of Bacillus amyloliquefaciens PMB01 to control bacterial leaf spot caused by Xanthomonas euvesicatoria on sweet pepper

林碩興(Shuo-Sing Lin)

指導教授 ： 林宜賢

國立屏東科技大學/農學院/植物醫學系所/碩士(2015年)

https://doi.org/10.6346/NPUST.2015.00029

摘要

茄科細菌性斑點病是甜椒生產的重要限制因子，此病害於田間主要利用銅劑防治，然而長期使用銅劑也導致田間的病原菌逐漸產生抗藥性。除了化學防治外，利用微生物來進行生物防治是最具潛力的防治策略，在利用微生物進行生物防治中，最常用的方法就是利用具有拮抗特性的微生物菌株來防治植物病害的發生。本研究中，首先由田間病葉分離甜椒細菌性斑點病病原，所獲得之PMXEU01菌株經生理生化測試、16S rDNA基因序列比對及應用專一性引子對，可確認此菌株為Xanthomonas euvesicatoria。以X. euvesicatoria為指示菌株，分析由土壤中分離之多個Bacillus菌株對此菌株在平板之拮抗能力。其中以Bacillus sp. PMB01菌株具有最好的拮抗能力。進一步將PMB01菌株利用16S rDNA和gyrB基因進行定序，均顯示此菌株為Bacillus amyloliquefaciens。在評估PMB01防治甜椒細菌性斑點病的可應用性之結果顯示，此菌株除在葉表殘存能力佳，至少可殘存21天，亦可降低細菌性斑點病的病害指數。在初步探討PMB01之可能發酵配方的結果顯示，利用不含黃豆粉的發酵配方所獲得之發酵液已能提高防治效果。同時，發酵濾液亦比nutrient broth培養濾液對PMXEU01具有較強的抑制作用，且此抑菌物質具耐熱的特性。在PMB01上亦可測得常見拮抗物質如iturin及surfactin產生的基因序列。由上述結果說明，發酵液具有較佳的防治效果可能與PMB01所產生的拮抗物質有關。除此之外，PMB01也具有甜椒葉表之纏據能力，也能在與甜椒細菌性斑點病病原菌共同存在下增加植物中H2O2的累積。綜上所述，本研究說明B. amyloliquefaciens PMB01可能藉由多種機制來防治甜椒細菌性斑點病。

關鍵字

茄科細菌性斑點病 ； 生物防治 ； Bacillus amyloliquefaciens ； 發酵

並列摘要

Bacterial leaf spot of sweet pepper caused by Xanthomonas spp. is an important limiting factor on fruit production. To control this disease, copper agents are commonly used in the fields. However, long-term usage of copper agents makes the bacterial pathogen become resistant. Therefore, we strive for an effective biocontrol methods. The causal agent of bacterial leaf spot were isolated from diseased sweet pepper. A strain, PMXEU01, was obtained and further identified as Xanthomonas euvesicatoria by physiological, biochemical assay and molecular identification. Then, the inhibitory effects of 11 Bacillus strains on PMXEU01 were assessed. Bacillus sp. PMB01 isolated from soil was the most effective strain to inhibit the growth of PMXEU01 on nutrient broth agar plate compared to other isolated strains. PMB01 was further identified as Bacillus amyloliquefaciens by sequences analysis of 16S rDNA and gyrB gene. In addition, PMB01 maintained its population on the leaves of sweet pepper for over three weeks. Thus, we further evaluated the biocontrol efficacy of PMB01 to X. euvesicatoria PMXEU01. Results showed the disease index caused by strain PMXEU01 was reduced with the treatment of B. amyloliquefaciens strain PMB01. To improve the biocontrol efficacy, fermentations of strain PMB01 in various media were accessed. Results revealed the biocontrol efficacy was improved by using fermentation media developed in this study. The cell free filtrates from fermentation liquid were heat stable and exhibited stronger inhibitory effect on X. euvesicatoria than that from nutrient broth. The existence of iturin and surfactin biosynthesis genes was found in strain PMB01. Thus, we suggested that the enhanced in antagonistic activity of strain PMB01 in fermentation liquid might be attributed to the increasing antimicrobial substances produced by PMB01 during fermentation. The strain PMB01 from both nutrient broth and fermentation liquid 1 (F1) exhibited well colonization ability on the leaves of sweet pepper. Moreover, strain PMB01 also intensified the ROS generation and ROS accumulation while the X. euvesicatoria PMXEU01 was presented in the leaves. We concluded that the biocontrol efficacy on bacterial leaf spot of sweet pepper might associate with multiple functions of PMB01. Therefore, we suggested that B. amyloliquefaciens PMB01 is a potential biocontrol agent to control bacterial leaf spot of sweet pepper.

並列關鍵字

Bacterial leaf spot ； biocontrol ； Bacillus amyloliquefaciens ； fermentation

參考文獻

9. 莊益源、周浩平、黃祥益、朱雅玲。2009。有機蔬菜綜合栽培管理技術。高雄區農技報導 95: 3-39。

6. 郭孚燿。2002。甜椒栽培。臺中區農業專訊 38: 19-24。

3. 吳雅芳、陳紹崇、彭瑞菊、黃淑惠、鄭安秀。2008。茄科細菌性斑點病病原細菌抗銅性之探討。臺南區農業專訊 64: 22-25。

1. 邱安隆。2004。植物病害生物製劑研發與應用。花蓮區農業專訊 48: 22-25。

12. 謝奉家。2011。臺灣芽孢桿菌生物殺菌劑的研發與應用現況。藥毒所專題報導 103: 1-3。