液化澱粉芽孢桿菌BPD1防治番茄青枯病之潛力

Potential of Bacillus amyloliquefaciens BPD1 for control of bacterial wilt of tomato

蘇靖雯(Ching-Wen Su)

指導教授 ： 劉嚞睿

國立臺灣大學/生物資源暨農學院/生物科技研究所/碩士(2017年)

https://doi.org/10.6342/NTU201702236

摘要

青枯病是由Ralstonia solanacearum所引起的土傳性病害，為台灣番茄栽培重要限制因子之一。青枯病菌能夠侵入植物根部組織，快速移動至維管束並散佈至全株，使罹病植物急速萎凋而死亡。台灣目前針對青枯病唯一核可的防治方法為施用生物防治菌。內生菌存在於植物組織中，較不受外在環境變化所影響，具有穩定的生存空間，因此近年來應用內生菌作為生物防治菌逐漸受到重視。先前研究已知Bacillus amyloliquafaciens BPD1（Ba-BPD1）能夠促進植物生長並具有拮抗多種植物病原菌的能力。本研究主要探討Ba-BPD1防治番茄青枯病的可能機制，以及是否能夠穩定存在於番茄組織及對植物內生菌相造成的影響。結果顯示Ba-BPD1培養於大豆配方培養基具有最佳抑制活性，無菌上清液能夠影響青枯病菌的移動性及細胞型態。分析Ba-BPD1的gDNA發現具有合成至少6種抑菌化合物的基因，包括bacillaene、macrolactin、difficidin、bacillomycin D、bacillibactinc和bacillycin。另外，Ba-BPD1產生的揮發性有機化合物亦能夠影響青枯病菌菌落生成大小、移動性及細胞型態。盆栽試驗中，前處理Ba-BPD1使聖女番茄青枯病罹病度下降39.6%。進一步探討其抗青枯病的可能作用機制，結果發現介質中青枯病菌數量並未受到Ba-BPD1的處理而顯著下降；另一方面，Ba-BPD1在聖女番茄受青枯病侵襲時，引起由茉莉酸和乙烯作為訊號分子的植物抗性；此外，連續施用Ba-BPD1三週能夠提升番茄之生質量。最後並證實Ba-BPD1能夠進入番茄組織中，同時造成番茄內生菌多樣性及結構改變。綜合上述結果，Ba-BPD1因能夠產生抑菌化合物、誘導植物系統性抗病、纏據於植物組織中，影響內生菌相組成並且促進植物的生長，故十分有潛力作為防治番茄青枯病的生物防治菌。

關鍵字

青枯病 ； 番茄 ； 生物防治 ； 液化澱粉芽孢桿菌 ； 內生菌 ； 誘導植物系統性抗性 ； 內生菌相

並列摘要

Bacterial wilt, which is caused by the soil-borne pathogen Ralstonia solanacearum (Rs), is one of the most serious diseases in tomato plant. Rs can invade plant roots and spread quickly into the xylem vessels toward the whole plant tissue through the vascular system. Symptoms of this disease include stunting, vascular browning, wilting, and even causing plant death. In Taiwan, the disease caused by Rs is managed only by using beneficial microorganisms. Nowadays, the study of endophytic bacteria in plants and their applications on biological control of plant pathogens are gaining a lot of interest. Bacillus amyloliquefaciens BPD1 (Ba-BPD1) has been shown to have plant-growth-promoting effects and the ability to produce several types of antimicrobial compounds against plant pathogens. In this study, we found that Ba-BPD1 has the biggest inhibition zone against Rs when incubated in LMS medium. Furthermore, after treatment with 5 μl/ml of the culture supernatant of Ba-BPD1, the Rs population decreased to 20% and their motility activity also decreased as well as the morphology of Rs cells. To identify the antimicrobial compounds probably present in the supernatant, six gene fragments of antimicrobial compounds, including bacillaene, macrolactin, difficidin, bacillomycin D, bacillibactinc, and bacillycin, were amplified from Ba-BPD1 through PCR analysis. Moreover, we also found that volatile organic compounds (VOCs) produced by Ba-BPD1 had negative effects on the colony size, morphology, and motility activity of Rs. In the pot experiments, we found that treatment with Ba-BPD1 could reduce the Rs disease severity by 39.6% in tomato cv. Santa. To further understand the mechanism of Ba-BPD1 inhibition of tomato plant wilting, we evaluated the pathogen populations in soil and the expression levels of the defense-related genes in leaf after Ba-BPD1 treatment. The results revealed that the soil pathogen populations were not significantly decreased after Ba-BPD1 treatment. On the other hand, gene expression levels of the AOC and ACO genes, which are involved in the JA and ET pathways, respectively, were increased after pathogen inoculation in Ba-BPD1 treatment. On the other hand, the growth of tomato seedling was influenced by Ba-BPD1 inoculation. Last but not the least, our experiments showed that Ba-BPD1 could colonize into the tomato plant and the populations reached up to 3.7×104 CFU/g tissue at the basal parts of the inoculation site (0-2 cm). Otherwise, composition of the endophyte community in root and diversity of the endophyte community in root and stem tissues changed in response to inoculation were correlated with tomato resistance to the disease. Thus, this study demonstrated that Ba-BPD1 has a potential to be used as a biocontrol agent in controlling tomato plant wilting caused by Rs as it is able to produce antimicrobial compounds, induce plant systemic resistance, colonize plant tissues in a stable manner, modulate the endophyte community of the host and also promote plant growth.

並列關鍵字

bacterial wilt ； tomato ； biocontrol ； Bacillus amyloliquefaciens ； endophytes ； induced systemic resistance ； endophyte community

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