多黏類芽孢桿菌TP3產生之有效抗菌物質分析

草莓灰黴病菌(Botrytis cinerea)是草莓栽培生產過程中的重要病害之一，由於草莓是連續採收作物，在這期間必須噴施化學農藥以減少果實因灰黴病而造成的損失，很難有安全採收期，一方面對環境造成汙染，及導致抗藥性菌株的產生。基於永續農業的發展，生物防治可以做為減少化學農藥使用的替代方案。生物防治的其中一個機制是利用微生物所產生之二次代謝物殺死或抑制病原菌，本實驗室自草莓植株葉圈分離到多黏類芽孢桿菌菌株(Paenibacillus polymyxa TP3)，可以有效抑制灰黴病菌生長，於溫室及田間試驗均能有效降低草莓灰黴病的罹病嚴度；另外也以本菌株防治草莓炭疽病，能在溫室試驗減緩炭疽病菌的感染，並能誘發草莓對炭疽病的抗性，說明菌株TP3 具有生物防治潛力。本研究主要分析菌株TP3 產生之有效抗灰黴病菌物質及其組成結構。利用生化分析之方法萃取菌株TP3 之抗菌物質，其粗萃物能有效抑制灰黴病菌之生長，進一步以高效液相層析儀並配合基質輔助雷射脫附游離飛行時間質譜儀(MALDI-TOF MS)偵測抗灰黴病菌分液之抗菌物質，推斷其為殺鐮孢菌素(fusaricidins)。此外，影像質譜儀(MALDI-imaging MS)顯示殺鐮孢菌素主要分佈在菌體周圍，同時在抑菌圈也有明顯的多黏菌素E 質譜訊號，於體外試驗發現其具有抑制灰黴病菌絲生長能力。最後以液相層析串聯質譜儀分析菌株TP3 殺鐮孢菌素之組成及結構，發現大部分與已知者相同，但其中質荷比(m/z) 869胺基酸組成之排列與相同質荷比之已知殺鐮孢菌素不同，而質荷比(m/z) 917 與已知(m/z) 931 在第一個胺基酸組成不同，推測為新穎的殺鐮孢菌素。

關鍵字

草莓灰黴病菌；多黏類芽孢桿菌；基質輔助雷射脫附游離飛行時間質譜儀；殺鐮孢菌素

並列摘要

Botrytis cinerea is one of the major pathogens in the cultivation of strawberry.Strawberry is a kind of continual harvest crop; spraying pesticides is necessary to reduce the loss caused by gray mold and difficult to have safety harvest period. Moreover,pesticides cause pollution in enviroments and emergence of drug-resistant strains. Based on the development of sustainable agriculture, biological control is proposed as an alternative to reduce the use of chemical pesticides. One of the mechanisms of biological control is the production of secondary metabolites to kill or inhibit the pathogens.Paenibacillus polymyxa strain TP3 isolated previously from the phyllosphere of strawberry could effectively inhibit the growth of B. cinerea, and in greenhouse and field trials, strain TP3 could effectively reduce the disease severity of strawberry gray mold. Furthermore, strain TP3 could retard the infection of Colletotrichum gloeosporioides in greenhouse experiment and induce strawberry resistance against anthracnose, illustrating the potential of strain TP3 as a biocontrol agent. In this research, the anti-B. cinerea compounds produced by strain TP3 and its composition and structure were investigated. By a biochemical approach, antifungal compounds which could effectively inhibit the growth of B. cinerea were extracted and analyzed. High performance liquid chromatography and matrix-asisted laser desorption ionization-time of flight mass spectrometry were applied to identify the composition of the anti-B. cinerea compounds, which was presumed to be fusaricidins. Moreover, matrix-asisted laser desorption ionization-time of flight-imaging mass spectrometry showed that fusaricidins mainly distributed around the bacterial colony. Simultaneously, it also found that polymyxin E appeared at inhibition zone with apparent mass signals and could inhibit B. cinerea growth in vitro. Finally, composition and structure of fusaricidins produced by strain TP3 were analysed by liquid chromatography-tandem mass spectrometry and the results indicated an identity of fusaricidins as previously reported; however, the amino acid sequence of m/z 869 was different from the known fusaricidin of same m/z and the first amino acid of m/z 917 was different from that of known m/z 931, suggesting to be a novel fusaricidin member.

並列關鍵字

Botrytis cinerea ； Paenibacillus polymyxa ； matrix-asisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) ； fusaricidins

參考文獻

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多黏類芽孢桿菌TP3產生之有效抗菌物質分析

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