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標題: 蕈狀芽孢桿菌防治番茄萎凋病之相關機制分析
Related mechanisms analyses for controlling tomato Fusarium wilt with Bacillus mycoides
作者: 湯佳蓉
Tang, Jia-Rong
Contributors: 黃振文
Jhen-Wun Huang
植物病理學系所
關鍵字: 番茄鐮孢菌萎凋病;誘導抗病;Bacillus mycoides
Tomato Fusarium wilt;induced resistance;Bacillus mycoides
日期: 2012
Issue Date: 2013-11-19 12:38:39 (UTC+8)
Publisher: 植物病理學系所
摘要: 在溫室利用 Bacillus mycoides CHT2402 及 NP02 細菌懸浮液分別拌入栽培介質後,培育番茄幼苗 14 天,再移植種於番茄萎凋病菌 (Fusarium oxysporum f.sp. lycopersici) 土中,結果發現兩菌株分別可降低植株罹病度 48.1% 及 46.2%。為了降低環境影響植株之抗病機制的表現,因此本研究於三角瓶中建立栽培番茄實生苗及接種生防菌及病原菌的技術,以了解生防菌誘導番茄植株抗病的相關機制。首先以 Biolog GP Microplate 分析番茄萎凋病菌 Fol-04 菌株及 B. mycoides CHT2402 和 NP02 兩菌株對碳、氮素源利用的需求,隨後比較生防菌及病原菌對營養需求的差異性,以便確立分析平台的基礎培養基配方,結果發現在 Murashige 和 Skoog 兩氏 (MS) 培養基中蔗糖的濃度調為 1% (w/v),最適合番茄植株、生防菌及病原菌等三者間的交互作用。在三角瓶與溫室栽培環境下,B. mycoides CHT2402 與 NP02 均可於處理 9 天後完全纏據番茄根部。將番茄種子於 CHT2402 及 NP02 菌液中催芽後培養於 MS 培養基二週,再以本病原菌單孢接種於番茄根部附近,5 天後發現對照組的植株已受病原菌感染且出現倒伏的現象,惟處理組植株尚維持挺立且未表現病徵。自三角瓶及溫室栽植處理及未處理過 B. mycoides CHT2402 及 NP02 菌株之番茄根部取樣後,以環氧樹脂包埋後切片,再以光學顯微鏡觀察它們的根組織之細胞結構,發現處理過 CHT2402 及 NP02 之植株根部於表皮細胞下方的細胞壁較對照組分別增厚 0.1-0.16 μm 及 0.12-0.17 μm,至於皮層細胞下方的細胞壁則是分別增厚 0.05-0.1 μm 及0.12-0.18 μm。利用穿透式電子顯微鏡觀察的結果,發現處理過 B. mycoides 番茄根部細胞大多於細胞間隙以非結晶物質累積於細胞壁,造成組織增厚的現象。在抗病基因表現方面,是於處理 B. mycoides 後第 9、11、13、15 天取樣後萃取其 mRNA 後,反轉錄成 cDNA 後以 qPCR 分析 PAL 及 LOX 基因的表現量,發現於三角瓶及溫室栽培環境中處理組植株的 LOX 與 PAL 基因均有被較高量誘導表現的現象。其中於三角瓶中處理 NP02 後第 11 天及處理 CHT2402 後第 13 天兩基因均有相同趨勢的高量表現;至於溫室中處理過 NP02 的番茄根部於處理後 11-13 天 PAL 基因有受誘導表現的現象且於處理後第 13-15 天 LOX 基因亦受誘導表現,而處理過 CHT2402的番茄根部則於處理後第 13 天僅 LOX 基因有受高量誘導表現的現象。綜合本研究結果証明 B. mycoides 須先於病原菌感染前纏據於番茄根部及維管束組織,並誘導番茄植株產生抗病反應,才能有效扺抗萎凋病菌的感染。
Bacillus mycoides CHT2402 and NP02 were effective in respectively reducing 48.1% and 46.2% disease severity of Fusarium wilt of tomato plants caused by Fusarium oxysporum f. sp. lycopersici Fol-04 in the greenhouse. In order to explore related mechanisms for inducing tomato plants resistant to Fusarium wilt disease by the biocontrol agent, a platform was set up for simultaneously culturing tomato seedlings, B. mycoides, and F. oxysporum f. sp. lycopersici Fol-04 in the flask cultivation system. Biolog GP Microplate was used to analyze the utilization of carbon and nitrogen sources by F. oxysporum f. sp. lycopersici Fol-04, B. mycoides CHT2402 and NP02. The results showed that sucrose concentration of Murashige’s and Skoog’s (MS) medium was adjusted to 1% (w/v) had more suitable interaction among tomato seedlings, the pathogen and biocontrol agents. The roots of tomato seedlings could be completely colonized by B. mycoides CHT2402 and NP02 nine days after the seeds were treated with cell suspension of the biocontrol agents and grown in the flask and greenhouse cultivation systems. Tomato seeds were incubated in the cell suspension (108 cfu/ ml) of B. mycoides CHT2402 and NP02 for 3 days, and then they were transplanted to the modified MS medium in the flask. Two weeks later, each of tomato seedlings was inoculated with single spore of F. oxysporum f. sp. lycopersici Fol-04 near the root. It was found that tomato seedlings could be protected from the pathogen by B. mycoides CHT2402 and NP02 for five days in the flask cultivation system. To study mechanisms for controlling tomato Fusarium wilt by the biocontrol agent, the root tissues of tomato plants treated with B. mycoides CHT2402 and NP02 were analyzed by tissue section and qPCR techniques. The results of Spurr’s resin block section indicated that the cell wall thickness of epidermis cells of tomato plant treated with B. mycoides CHT2402 and NP02 did increase 0.1-0.16 μm and 0.12-1.17 μm, and the cell wall thickness of cortex cells increased 0.05-0.1 μm and 0.12-0.18 μm. In addition, the results of qPCR showed that expression of LOX and PAL genes of tomato plant were induced by B. mycoides CHT2402 and NP02 in both cultivation conditions. According to above results, it was found that B. mycoides CHT2402 and NP02 were able to control tomato Fusarium wilt if they could colonize the roots and vascular tissues of tomato plants prior to the pathogen infection.
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