篩選具抑制花生根瘤線蟲(Meloidogyne arenaria)的內生細菌以開發成新穎之生物農藥

Screening for Novel Biocontrol Endophytic Bacteria that Inhibit Root-Knot Nematode (Meloidoyne arenaria)

袁華靖(Hua-Jing Yuan)

指導教授 ： 楊禮亘

國立虎尾科技大學/文理學院/生物科技系碩士班/碩士(2016年)

摘要

植物寄生性根瘤線蟲普遍存在於氣候炎熱或冬天短暫的土壤中，約有2000多種植物易受到根瘤線蟲的感染並造成全球農業巨大的損失。目前已知根瘤線蟲種類有七十多種，其中下列四種為台灣最常見且危害農作物較嚴重的根瘤線蟲：(1)花生根瘤線蟲；(2)爪哇根瘤線蟲；(3)南方根瘤線蟲；(4)北方根瘤線蟲。植物感染根瘤線蟲後其根部會形成許多的腫瘤(根瘤)，影響植物水分及養分的吸收、改變植物生理生化代謝、減少葉綠素含量等，嚴重則會導致植物的死亡。針對根瘤線蟲的防治方法目前管理主要是倚賴化學性殺線蟲藥劑，然而許多殺線蟲劑為致癌物質，除了因使用不當而殘存農業產品中影響人體健康外，土壤及地下水的污染亦會造成環境汙染。因此，許多國家已禁止使用殺線蟲劑而尋求更良性和環保的生物防治對策。為尋求生物性替代製劑，本研究利用已建立之水稻、玉米、十字花科蔬菜、藥用植物等作物內生菌種資源庫以篩選具有抑制根瘤線蟲能力之抑線菌，並進一步以接種方式與常用殺線蟲劑為對照測試其功效。結果發現抑線菌LS012 (Xanthomonas sp.)能顯著抑制花生根瘤線蟲卵的孵化率達94%，而二齡幼蟲的致死率72%。經進一步接種空心菜實驗發現，此菌株不影響植物之生長發育且在分別預先接種菌體及菌上清液實驗組中發現上清液具無感染內生菌控制組較強之抑制線蟲病徵能力，植株之根瘤指數為2.33而控制組為3.67。以管柱層析發現菌上清液中的第6分割段含有抑制線蟲卵塊孵化之生物活性分子。本研究之初步結果及所建立之微生物抑制線蟲篩選平台可提供未來根瘤線蟲生物農藥製劑之開發。

關鍵字

根瘤線蟲 ； 內生細菌 ； 生物農藥

並列摘要

Plant parasitic nematodes exist in hot climate or short winter soil areas and there are about 2000 plant species that are susceptible to root-knot nematode infection, which cause huge global crop losses. There are about seventy different species of root-knot nematode, whereas the following four are the most common and cause serious crop damages in Taiwan: (1) Meloidogyne arenaria; (2) Meloidogyne javanica; (3) Meloidogyne incognita; (4) Meloidogyne hapla. Root-knot nematode infection can lead to the formation of root nodules, thereby affecting the absorption of water and nutrients, changes in plant physiology and biochemical metabolism, reduce chlorophyll content, and even result in death of the plant. Present control methods for root-knot nematode infestation mainly rely on chemical nematocides, yet, this method is high in cost or often inefficient, and also likely to cause environmental pollution. In addition, residual chemicals may affect human health and thus are banned in many countries. Therefore, the search for a more environmental friendly and acceptable biological control approach is a global research priority today. This study is based on the endophytic bacterial collection from rice, corn, cruciferous vegetables and medicinal plants to screen for endophytic bacteria that inhibit the hatching of root-knot nematode, and further compared with the commonly used nematocides as a control to test their efficacy. The results showed Xanthomonas sp. strain LS012 could significant inhibit the hatching of root-knot nematode eggs (94% inhibition). After further inoculation in spinach, we found that this strain does not affect the growth and development of plants. Pre-inoculation of LS012 or its supernatant on spinach showed the supernatant exhibited strong inhibition of nematode symptoms in regards to plant vigor and number of nodules (RKI=2.33 as compared to RKI=3.67 in uninoculated control). Column chromatography revealed the 6th fraction of the effluents contains biologically active molecules that suppress nematode egg hatching. The preliminary work and the establishment of the microorganism-nematode screening platform can provide us for the development and application of biological control agents for nematode management in the future.

並列關鍵字

Root-knot nematode ； Endophyte ； Biological control agents

參考文獻

4. 吳雅芳。2014。台南農業專訊89期。行政院農業委員會台南區農業改良場發行。18-20。

10. 張上鎮、王升陽、葉丁峰、吳季玲。1997。超音波法快速萃取及定量葉綠素。台灣林業科學，12：329-334。

17. 蔡東纂。1996。台灣作物線蟲病連作障害之發生及對策。植物病理學會刊，5：113-128。

84. Siripak, T., Tsai, T.T., Chen, P. 2013. Identification of root knot nematode Meloidogyne species on rice in Taiwan. Proceedings of 2013 Annual Meeting of Taiwan Phytopathological Society：53-54.

11. 陳任芳。2009。花蓮區農業專訊68期-農業新科技與新知專輯。行政院農業委員會花蓮區農業改良場發行，14-16。