利用膜輔助策略開發有效篩選促進植物生長根棲細菌之方法

Development of an Efficient Method for Screening of ‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬Plant Growth Promoting Bacteria Using a Membrane-Assisted Strategy

大矢裕之(Hiroyuki Oya)

指導教授 ： 林乃君

共同指導教授 ： 青柳秀紀

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201900535

摘要

促進植物生長細菌 (plant growth-promoting bacteria, PGPB) 已被廣泛認知可直接或間接地促進植物生長。因此，透過分離後利用數種耗工的培養方法篩選菌株之製造螯鐵蛋白、溶磷和合成 IAA 等促進植物生長特性 (plant growth-promoting traits, PGPT)，便可獲得許多具潛力的PGPB。雖然並非所有具 PGPT 的細菌都有促進植物生長的效果，但以上述傳統方式評估隨機從土壤和根圈等環境中篩選出來的眾多細菌，常可有效篩選出潛力菌株。儘管此為重要議題，但針對提高傳統方法的效率，也就是能更快篩選到具有較高促進植物成長能力微生物的研究仍佔少數。此外，僅使用單一培養基自一般或根圈土壤分離培養菌株的方法，可能會因未使用對的培養條件而造成可培養的 PGPB 數量與種類是有限的。因此，本研究中擬使用各種纖維素薄膜發展 (1) 新穎且更有效評估 PGPT 的方法，以及 (2) 培養更多樣化微生物群落種類的方法。首先在台灣以傳統方式選擇 5 株具有高 PGP 潛力的 PGPB，並進行番茄共培養試驗，評估其促進植物生長的能力。接下來，便以此些菌株進行測定製造螯鐵蛋白、溶磷和合成 IAA能力方法的優化；結果發現，與傳統方式比較，(1) 搭配使用超純纖維膜 (ultrapure cellulose sheet, UCS) 和硝化纖維膜 (nitrocellulose membrane, NC membrane) 的系統有更好的表現；(2) 在平板上放置 UCS、SCF 或 NC membrane 使培養基表面結構改變，及改變培養基成分兩方法，均可培養出不同的微生物群落。因此，這些新開發的評估方法可能有助於讓我們在未來獲得更多且更多樣化的 PGPB。

關鍵字

促進植物生長細菌 ； 製造螯鐵分子 ； 溶磷 ； 合成 IAA ； 膜輔助策略 ； 無法培養之微生物

並列摘要

It’s widely known that plant growth-promoting bacteria (PGPB) can assist plant growth directly or indirectly. For that reason, many potential PGPB were obtained through isolation followed by screening for their plant growth-promoting traits (PGPTs) such as Indole-3-acetic acid (IAA) production, siderophore production and phosphate solubilization using several labor-consuming, cultivation-dependent assay methods. However, even though bacterial strains with PGPT identified using the conventional assay method do not always possess the ability to promote plant growth, evaluation of bacteria collected from soil or rhizosphere using traditional screening methods is always required. Although this is an important issue, only a few researches have been carried out from this perspective. Furthermore, numbers or species of bacteria isolated from suspension made of bulk or rhizosphere soil using only one medium might be limited as specific cultivation conditions might be required for certain unculturable micro-flora in the environment samples. Hence, in this study, (1) development of a novel, efficient ‬ ‪assay method and (2) diversifying unculturable micro-flora in conventional methods by using cellulose films were attempted. Initially, 5 potential PGP isolates with high PGP activities from Taiwan were selected using a conventional method with the assay media, and their in planta PGP activities on tomato plants were also evaluated. Then, isolates were used to normalize the procedure to check three PGPTs, including siderophore production, phosphate solubilization and IAA synthesis. The results showed that (1) better performance can be obtained using a continuous membrane-assisted PGPT assay with nitrocellulose (NC) membrane and ultrapure cellulose sheet (UCS) compared to the conventional method, and (2) changes in both surface structure/architecture of the agar plate with UCS, specific cellulose film (SCF), and NC membrane on it and medium composition can give rise to different cultivable bacteria compared to the conventional method. These newly developed assay methods could allow us to isolate more abundant and diversified PGPB in the future.

並列關鍵字

Plant growth-promoting bacteria ； Siderophore production ； Phosphate solubilization ； IAA synthesis ； Membrane-assisted strategy ； Unculturable microorganisms

參考文獻

Abadía, J., Lopez-Millan, A. F., Rombola, A. & Abadia, A. (2002).: Organic acids and Fe deficiency: A review. Plant and Soil, 241: 75–86.

Ajmal, M., Ali, H. I., Saeed, R., Akhtar, A., Tahir, M., Mehboob, M. Z. & Ayub, A. (2018).: Biofertilizer as an alternative for chemical fertilizers. RRJAAS, 7: 1–7.

Aksorn, E. & Chitsomboon, B. (2013).: The Effects of Plant Growth Promoting Traits on Heavy Metal Uptake of Vetiver Grasss. Am.-Euras. J. Agric. Environ. Sci., 13: 465-470.

Alexander, D. B. & d Zuberer, D. A. (1991).: Use of chrome azurol S reagents to evaluate siderophore production by rhizosphere bacteria. Biol. Fertil. Soils, 12: 39– 45.

Ali, S., Charles, T. C. & Glick, B. R. (2017).: Endophytic phytohormones and their role in plant growth promotion. Funct. Impt. Plant Microbiome. 7: 89–105.