建立Bacillus amyloliquefaciens PMB04的液態發酵配方於甜椒細菌性斑點病之防治

甜椒是世界上重要作物之一，在其栽培過程中由Xanthomonas perforans 所造成的細菌性斑點病會造成品質及產量嚴重降低。除了化學防治方法之外，開發可防治甜椒細菌性斑點病之微生物製劑也有其必要性。本研究擬應用已證明對十字花科黑腐病及草莓炭疽病具有良好防治效果之Bacillus amyloliquefaciens PMB04，評估其在防治甜椒細菌性斑點病之潛力並建立其發酵液配方。在平板拮抗活性分析的結果顯示B. amyloliquefaciens PMB04可抑制多種病原細菌之生長，其中又以對X. perforans 之拮抗效果最為顯著。進一步利用B. amyloliquefaciens PMB04之細菌懸浮液進行處理，亦可顯著降低甜椒之細菌性斑點病發生。為了提升B. amyloliquefaciens PMB04在田間應用的潛力，以PMBFL-2A作為基礎發酵配方，分別利用搖瓶試驗及30公升發酵槽，以不同比例之碳氮比進行配方調整。在菌量及產胞率的結果顯示，於搖瓶發酵中以碳氮比0.5-0.5及1-1之配方發酵後可達100%之產胞率；在30公升發酵槽中以碳氮比3-1、2-1以及0.5-0.5之配方均可達100%之產胞率。將前述由30公升發酵槽所獲得之發酵液進行防治試驗結果顯示，以碳氮比0.5-0.5所製備之發酵液配方(PMB4FL)可達44.5%之防治率為最佳，接著將PMB4FL發酵液進行200、500及1000倍稀釋處理也能顯著減少病害的發生，防治率分別為39.1%、28.7%及16.8%，且將PMB4FL發酵液於54 °C下存放14天仍可維持100%之存活率。進一步評估茄科作物常用之殺菌劑搭配B. amyloliquefaciens PMB04之可行性，由液態培養的結果顯示，包括含銅殺菌劑在內的部分殺菌劑對B. amyloliquefaciens PMB04的生長略有抑制，表示未來施用B. amyloliquefaciens PMB04發酵液時需避免與殺菌劑混合使用。同時在含銅殺菌劑與X. perforans之拮抗分析的結果顯示，氫氧化銅對於本實驗之供試菌株具有較穩定之抑制效果，因此進一步分析氫氧化銅對PMB4FL發酵液於甜椒細菌性斑點病防治效果的影響，結果顯示氫氧化銅無法增強PMB4FL發酵液對甜椒細菌性斑點病之防治效果外，單獨處理氫氧化銅亦無防治效果，由此說明B. amyloliquefaciens PMB04發酵液在甜椒細菌性斑點病之防治上以單獨處理為佳。最後，本研究亦分別利用antiSMASH軟體、PCR及薄層層析進行分析後，推測B. amyloliquefaciens PMB04可產生iturin、fengycin及bacilycin等拮抗物質，並進一步利用B. amyloliquefaciens PMB04之濾液探討其對X. perforans XL1之影響，結果顯示PMB4FL發酵液濾液可更為顯著抑制X. perforans XL1之細菌族群量。綜上所述，本研究建立之PMB4FL配方，可使B. amyloliquefaciens PMB04具有最高產胞率且單獨處理即可顯著降低甜椒細菌性斑點病之發生，具有實際應用於田間的潛力。

關鍵字

細菌性斑點病；液態發酵；殺菌劑；拮抗物質

並列摘要

Sweet pepper is an important vegetable crop worldwide. In the production process of sweet pepper, bacterial leaf spot (BLS) caused by Xanthomonas perforans has serious impact on quality and yield. In addition to chemical control, it is necessary to develop a biocontrol agent that can control BLS on sweet pepper. This study is aim to apply Bacillus amyloliquefaciens PMB04, which could control crucifer black rot disease and strawberry anthracnose, to evaluate its potential o on control BLS and further establish the liquid fermentation formula of B. amyloliquefaciens PMB04. First, the inhibitiory assay revealed that B. amyloliquefaciens PMB04 has strongest antagonistic activity against several bacterial pathogens, especially on X. perforans. Further apply the bacterial suspension of B. amyloliquefaciens PMB04, result revealed that the disease severity was significant decrease on sweet pepper. To increase its application potential in field, the PMBFL-2A was used as the basal formula, and further adjusted the different ratios on carbon and nitrogen in the 500 ml flask and 30 liters fermentation tank. Results exhibited that bacterial population and sporulation in the formula with carbon and nitrogen ratio at 0.5-0.5, named PMB4FL, had best sporulation to 100% in both 500 ml flask and 30-liter fermentation tank. The PMB4FL fermentation broth has 100% survival rate under 54 °C for 14 days. And, the biocontrol efficacy to 44.5% of PMB4FL was best. Subsequently, the control efficacies reduced by PMB4FL fermentation broth at 200, 500 and 1000-fold dilutions were 39.1, 28.7 and 16.8%. Further evaluated the inhibition effect of fungicides suggested for controlling disease of Solanaceae on growth of B. amyloliquefaciens PMB04, the result revealed that part of fungicides including copper based-fungicides could suppress the growth of B. amyloliquefaciens PMB04. These results indicated the fermentation broth of B. amyloliquefaciens PMB04 should be avoid of using with fungicides. In addition, the result revealed copper (II) hydroxide could suppress bacterial growth of X. perforans strains. However, the co-treatment of copper (II) hydroxide and PMB4FL fermentation broth, copper (II) hydroxide did not increase the biocontrol efficacy of PMB4FL fermentation broth to BLS on sweet pepper. Besides, the treatment with copper (II) hydroxide did not exhibit any control efficacy to that. The metabolites anlysis performed by genome prediction, PCR and thin-layer chromatography assay suggested that B. amyloliquefaciens PMB04 might have iturin, fengycin and bacilysin synthesis. And, the filtrate of PMB4FL ferementation broth was reduce the population of X. perforans XL1 significantly. Taken together, B. amyloliquefaciens from PMB4FL liquid fermentation formula has highest sporulation and decrease disease severity of bacterial leaf spot on sweet pepper effectively. Application of the fermentation broth alone to control BLS would be promising in the field.