綠葉假單胞菌作為香蕉黃葉病生物防治菌之探討

Pseudomonas chlororaphis as a potential biocontrol agent against Panama disease caused by Fusarium oxysporum f. sp. cubense

施媞佳(Lyania Sartika)

指導教授 ： 沈偉強

共同指導教授 ： Liliek Sulistyowati ； Luqman Qurata Aini(Luqman Qurata Aini)

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202100714

摘要

香蕉黃葉病，由病原真菌 Fusarium oxysporum f. sp. cubense（Foc）所引起，為常見的土傳性病害，造成香蕉萎凋病徵。此病害分布廣泛，並以東南亞地區最為普遍，對全球香蕉的生產具有重大威脅。香蕉黃葉病菌可透過罹病組織、人類移動，及灌溉水等途徑傳播，病原菌可藉由厚膜胞子長時間殘存於土壤中。現行香蕉黃葉病的防治策略，包括種植抗病蕉種、施用農藥，及田間衛生管理耕作防治等，其中生物防治菌的施用，咸信是對環境友善及產業永續的另一防治策略。
在本實驗室前人的研究中，從有機田區香蕉根部分離出的假單胞菌屬（Pseudomonas） 菌株 AH1E1，對香蕉黃葉病菌熱帶第四型生理小種（TR4）展現生物防治的潛力。假單胞菌屬細菌為已知具有潛力的生物防治菌，具有促進植物生長、抑制病原菌，及誘導植物抗性等能力。本研究進一步利用atpD、carA、recA、16s rRNA、gyrB、rpoB，及rpoD等基因序列片段及全基因體資訊，進行比對與親緣分析，確定AH1E1菌株的分類地位為 Pseudomonas chlororaphis subsp. aurantiaca。生化測定包括螯鐵蛋白（Siderophore）、吲哚乙酸（Indole acetic acid）、氧化酵素活性，以及溶磷能力等，顯示 AH1E1 具有促進植物生長及生態適應等特性。進一步利用液相層析質譜儀（LC MS/MS），分析AH1E1與Foc TR4的對峙培養樣品，發現 pyrrolnitrin及pyochelin的存在。AH1E1菌株也進一步進行全基因定序，完整組裝顯示基因體大小為7,343,089鹼基對，並找到pyrrolnitrin 和pyochelin 生合成的兩個操縱組。透過綠色螢光蛋白標定菌株進行定殖觀察，顯示AH1E1能於香蕉根圈及部分根部組織進行定殖纏據。此外，溫室盆缽生物防治試驗結果顯示，AH1E1能有效抑制黃葉病的發生。未來期望能開發劑型及施用流程，進一步測試及應用Pseudomonas chlororaphis AH1E1於田間黃葉病的防治，提升香蕉產業的永續發展。

關鍵字

香蕉黃葉病菌 ； 香蕉黃葉病 ； 綠葉假單胞菌 ； 生物防治菌 ； 硝吡咯菌素 ； 螯鐵蛋白

並列摘要

Panama disease caused by Fusarium oxysporum f. sp. cubense (Foc) is a common soil-borne disease that causes wilt in banana plants. This disease is widespread, especially in Southeast Asia, and has been threatening worldwide banana production and productivity. The pathogen is transmitted through infected plant materials, human movement, and irrigation water, and the survival structure of Foc, chlamydospore, can remain in soils for a long period. Management strategies such as the use of resistant cultivars, fungicides, field sanitation, and other cultural practices have been suggested for Panama disease. Among them, the implementation of biocontrol agents is an ecologically friendly and promising way for sustainable banana production. Previous studies in the laboratory isolated Pseudomonas sp. AH1E1 from root tissues of bananas grown in organic fields, which shows promising biocontrol ability against Foc tropical race 4 (TR4) strain. Pseudomonas spp. as potential biocontrol agents have been demonstrated to contain various abilities including plant growth promotion, suppression of pathogenic microorganisms, and induction of plant resistance. In this study, Pseudomonas sp. AH1E1 was further identified as Pseudomonas chlororaphis subsp. aurantiaca based on the sequences of several housekeeping genes (atpD, carA, recA, 16s rRNA, gyrB, rpoB, and rpoD genes) and genome phylogeny study. Biochemical assays for the siderophore, indole acetic acid, oxidase, and phosphate solubilization activities demonstrated this strain exhibited various traits for plant growth promotion and ecological fitness. Chemical analysis by LC-MS/MS was conducted to screen for potential antifungal compounds produced by P. chlororaphis AH1E1 against Foc TR4 in dual culture assay. Pyrrolnitrin and pyochelin were found to be present in the fractions. This strain was further completely sequenced and the genome size was 7,343,089 bp in length. Two operons, potentially responsible for the biosynthesis of pyrrolnitrin and pyochelin, were also identified from the genome. Colonization study using GFP-tagged strain showed this strain efficiently colonized the rhizosphere of banana roots and also grew limitedly inside root tissues. Pot biocontrol assay showed that P. chlororaphis AH1E1 can efficiently suppress the disease under laboratory conditions. In the future, we hope to further develop a formulation and protocol of this candidate bacterium for field application to sustain banana production.

並列關鍵字

Fusarium oxysporum f. sp. cubense ； Panama disease ； Pseudomonas chlororaphis ； biological control agent ； pyrrolnitrin ； pyochelin

參考文獻

Abd-El-Khair, H., Elshahawy, I., and Haggag, H. K. 2019. Field application of Trichoderma spp. combined with thiophanate-methyl for controlling Fusarium solani and Fusarium oxysporum in dry bean. Bull. Natl. Res. Cent. 43:19.

Ahmad, F., Ahmad, I., and Khan, M. 2008. Screening of free-living rhizospheric bacteria for their multiple plant growth promoting activities. Microbiol. Res. 163:173-181.

Akila, R., Rajendran, L., Harish, S., Saveetha, K., Raguchander, T., and Samiyappan, R. 2011. Combined application of botanical formulations and biocontrol agents for the management of Fusarium oxysporum f. sp. cubense (Foc) causing Fusarium wilt in banana. Biol. Control 57:175-183.

Ajilogba, C. F., Babalola, O. O., and Ahmad, F. 2013. Antagonistic effects of Bacillus species in biocontrol of tomato Fusarium wilt. Stud. Ethno-Med. 7:205-216.

Alabouvette, C., Chantal, O., Quirico, M., and Christian, S. 2009. Microbiological control of soil-borne phytopathogenic fungi with special emphasis on wilt-inducing Fusarium oxysporum. New Phytol. 184:529-544.