光桿菌Photorhabdus akhurstii防治草莓炭疽病產生之有效物質特性分析

Abstract

草莓是一高產量且高經濟價值的作物，臺灣主要栽培草莓地區為苗栗縣大湖鎮，在栽培過程中如遇上高溫高濕度之環境會引發草莓炭疽病(Strawberry anthracnose)；主要是由Colletotrichum gloeosporioids (Cg) 病原菌所造成，會造成草莓經濟及收成有極大的損失和影響。在過去的病害防治上，多使用化學農藥來進行防治，然而過度的使用會導致抗藥性產生、食安問題以及藥劑對農民健康的危害。因此期望生物農藥可以成為替代的方案。本研究首先在對峙平板上確立了液化澱粉芽孢桿菌(Bacillus amyloliquefaciens) ASB111和光桿菌第一型(Photorhabdus akhurstii sp. nov. 0813-124 phase I) PL1有顯著的抑制效力，且PL1對其它Colletotrichum屬的病原菌也具有防治效力。基於研究的潛力及PL1防治草莓炭疽病的機制尚未明確，PL1成為我們主要進行研究的對象。在建立穩定的培養條件實驗中，得知PL1在經過24小時培養的缺鐵培養基(LP)中有最高的抑制效力，然而PL1在PDB液態培養基中未能被培養出。有趣的是，PDB agar (PDA) 在固態培養基的選擇試驗上顯示能使PL1產生有效物質去抑制Cg。另外，在最佳的萃取條件實驗中，顯示以乙酸乙酯(EA)萃取挖下的agar菌盤能獲得較高效率的粗萃物。經由高效液向層析儀(HPLC)分離純化PL1乙酸乙酯粗萃物，再透過液相質譜儀與串聯式質譜儀獲得之MS1與MS2數據進行分析，整合了二次質譜碎片進行結構預測，得到主要的抗生物質為glidobactin A, cepafungin I 和glidobactin C，它們均屬於syrbactins家族的化合物。除此之外，根據一次質譜的層析圖，這類的有效物質會受到agar固態培養的方式被誘導產生，且PDA能誘導比LPA更多的有效物質。利用質譜儀所得之結果結合全基因組定序；在全基因組定序經由antiSMASH的分析結果，PL1和PL2都有22個生合成基因簇，第8基因簇中有26%的基因與glidobactin具有相似、第15基因簇中也有15%的基因與glidobactin具有相似，我們猜測這可能與glidobactin的生合成路徑有關，然而這些生合成基因尚需進一步的實驗加以驗證。

Strawberries is an important crop and generates substantial economic values for farmers in Taiwan. Colletotrichum gloeosporioides (Cg) is one of the main pathogens in the cultivation of strawberry, which is also called strawberry anthracnose. This disease causes severe loss in strawberry production. Spraying the pesticides is a general practice to reduce the loss caused by anthracnose in the field. However, overuse of the chemical pesticides brings many problems such as the pollution in environments, emergence of drug-resistant strains, food safety risks and the health of the farmers. So, biological control is proposed as an alternative approach to replace chemical pesticides. In this study, the antagonistic assays showed that Bacillus amyloliquefaciens strain ASB111 and Photorhabdus akhurstii sp. nov. 0813-124 phase I (PL1) had anti-Cg ability. In addition, PL1 also had the ability against several other Colletotrichum spp. PL1 was chosen for further analyses to identify novel anti-Cg compounds and to elucidate the mechanisms for PL1 against Cg. PL1 grown in the iron limited medium (LP) at the first 24hr culture time had the highest inhibitory activity, and PL1 could not grow in potato dextrose broth (PDB). Interestingly, PL1 was induced to produce active compounds in PDB agar (PDA). Among different extraction methods, the cut-agar method and EA extraction had the best extraction efficiency. The active compounds of EA extracts were purified by high-performance-liquid-chromatography (HPLC) and identified by high resolution and high mass accuracy experiments (Mass spectrometry). By integrating MS spectra, MS fragments and structure prediction, the active compounds of PL1 were identified as glidobactin A (m/z 521), cepafungin I (m/z 535) and glidobactin C (m/z 549), all of which belong to the syrbactin family. Base on MS spectra, active compounds of syrbactin family could be highly extracted in the cut-agar extraction. The active compounds might be induced by the incubation on the PDA. In addition, PL phase II (PL2) did not have anti-Cg ability. According to the anti-SMASH analysis of whole genome sequencing, PL1 and PL2 had 22 biosynthetic gene clusters (BGCs). Among the clusters, region 8 showed 26% of genes were similar to glidobactin and region15 showed 15% of genes were similar to glidobactin. Thus, we hypothesize that the different antagonistic abilities of PL1 and PL2 might be caused by differential expression of genes in the syrbactin family biosynthetic pathway.