蕈狀芽孢桿菌防治番茄萎凋病之機制分析平台

The analysis platform for mechanisms on controlling tomato Fusarium wilt with Bacillus mycoides

湯佳蓉(Tang, J. R.) ； 張碧芳(Chang, P. F. L.) ； 張道禾(Chang, T. H.) ； 林盈宏(Lin, Y. H.) ； 黃振文(Huang, J. W.)

《植物醫學》 61卷1期 (2019/03) Pp. 29-38

https://doi.org/10.6716/JPM.201903_61(1).0003

摘要

本研究在三角瓶中栽培番茄實生苗與接種生物防治菌及植物病原菌，以分析生物防治菌誘導番茄植株產生抗病的相關機制。以Biolog GP Microplate分析番茄萎凋病菌Fusarium oxysporum f. sp. lycopersici Fol－04菌株及Bacillus mycoides CHT2402和NP02兩菌株對於碳、氮素源的需求，隨後比較生物防治菌及病原菌對營養需求的差異性，以便確立基礎培養基的配方，結果得知在Murashige和Skoog兩氏（MS）培養基中蔗糖的濃度調為1%（w/v），最適於番茄植株、生物防治菌及病原菌等三者間的交互作用。將番茄種子於B. mycoides CHT2402與NP02菌液中催芽後培養於MS培養基二週，再以番茄萎凋病菌單孢接種於番茄莖基部，5天後可見對照組的植株已受病原菌感染且出現倒伏的現象，惟兩生物防治菌處理過的植株尚維持挺立且未表現病徵。自三角瓶、溫室栽植處理及未處理過B. mycoides CHT2402與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根部組織之細胞結構，可見處理過B. mycoides CHT2402與NP02之植株根部於表皮細胞下方的細胞壁均較對照組分別增厚0.015－0.018 μm與0.014－0.016 μm以上，至於皮層細胞下方的細胞壁也有是分別類似增厚的現象0－0.008 μm與0.014－0.03 μm。利用穿透式電子顯微鏡觀察的結果，顯示處理過B. mycoides之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。在防禦相關基因表現方面，以即時定量反轉錄聚合酶鏈鎖反應（real time quantitative reverse transcription－polymerase chain reaction，real time qRT－PCR）分析處理B. mycoides之番茄根部於第9、11、13、15天後之苯丙氨酸氨裂解酶（phenylalanine ammonia lyase，PAL）與脂氧合酶（lipoxygenase，LOX）基因表現差異。結果顯示於三角瓶中處理B. mycoides NP02後第11天與處理B. mycoides CHT2402後第13天PAL與LOX基因有提高表現之趨勢；另外，溫室中處理過B. mycoides NP02的番茄根部於 11－13天後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；然而處理過B. mycoides 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。綜合本研究結果得知B. mycoides須先在病原菌感染前纏據於番茄根部與維管束組織，且誘導番茄植株產生防禦反應，才能有效扺抗萎凋病菌的感染。

關鍵字

番茄鐮孢菌萎凋病 ； 誘導抗病 ； 蕈狀芽孢桿菌

並列摘要

In order to explore the control mechanisms for tomato plants resistant to Fusarium wilt disease by the biocontrol agent Bacillus mycoides, a platform was set up for simultaneously culturing tomato seedlings, B. mycoides, and Fusarium 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 isolates. The results showed that adjusting sucrose concentration in Murashige's and Skoog's (MS) medium to 1% (w/ v) was suitable for the interactions among tomato seedlings, the pathogen, and biocontrol agents. Tomato seeds were incubated in the cell suspension (10^8 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 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 the mechanisms for controlling tomato Fusarium wilt by the biocontrol agents, the root tissues of tomato plants treated respectively with B. mycoides CHT2402 and NP02 were analyzed by tissue section and real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) techniques. The results of Spurr's resin block section indicated that the cell wall thickness of epidermis cells of the tomato plant treated with B. mycoides CHT2402 and NP02, respectively, increased 0.015-0.018 μm and 0.014-0.016 μm, whereas the cell wall thickness of cortex cells increased 0-0.008 μm and 0.014-0.03 μm. In addition, the expression of PAL (phenylalanine ammonia lyase) and LOX (lipoxygenase) genes in tomato roots was analyzed by qRT-PCR after B. mycoides CHT2402 and NP02 application in both flask cultivation and greenhouse condition. Accordingly, the results demonstrated B. mycoides CHT2402 and NP02 application induced the expression of PAL and LOX in tomato plants at different time points. Collectively, our results suggest that B. mycoides CHT2402 and NP02 are able to control tomato Fusarium wilt if they could colonize the roots and vascular tissues of tomato plants and trigger PAL and LOX expression prior to pathogen infection.

並列關鍵字

tomato Fusarium wilt ； induced resistance ； Bacillus mycoides