根瘤線蟲電漿激活水防治系統之開發與運用

Abstract

根瘤線蟲 ( Root-knot nemetodes, Meloidogyne spp. ) 為具有全球經濟重要性的植物寄生性線蟲。其寄主範圍廣泛，會使寄主根系形成瘤狀物，影響根系發展，使作物生長不良、葉片黃化、植株矮小等問題，影響農作物之品質與產量。多年來，農民倚賴施用殺線蟲藥劑對根瘤線蟲進行防治。由於殺線蟲劑大多具有神經毒的特性，危害人類健康與影響環境甚鉅，近年來，隨農業永續經營意識提升，數種殺線蟲劑已遭到禁用。剩餘可用的有限藥劑中，許多具有相同作用機制，導致田間線蟲族群已逐漸有抗藥性產生，防治效果不佳。因此，開發新型線蟲防治技術，迫在眉睫。電漿 ( Plasma ) 是自然界中的第四態為游離化的氣體，將其通入水中可製備成電漿激活水 ( Plasma activated water )。有鑒於相關研究指出其對微生物的抑制效果，本研究探討電漿激活水對防治根瘤線蟲之效能及運用之可行性。 本研究以彰化縣溪州鄉所採集的根瘤線蟲為實驗目標，並以 DBD ( Dielectric barrier discharge ) 方式製備的電漿激活水為資材，測試其對線蟲之僵直效果，與盆栽環境中之不同稀釋濃度的電漿激活水對線蟲的預防與治療效果。首先，經陰部膜紋形態鑑定和 PCR 分子及基因序列比對，鑑定出目標線蟲為象耳豆根瘤線蟲 ( Meloidogyne enterolobii ) 以及南方根瘤線蟲 ( Meloidogyne incognita ) 。實驗系統透過以氣體帶動電漿使其通入 ddH2O 的設計，製備成 pH 值為 3、電導度約 300 μs/cm 和氧化還原電位為 580 mv 的電漿激活水。本研究透過擊昏實驗發現，將二齡幼蟲浸泡於稀釋至 75 % 電漿激活水中 2 個小時，能造成 100 % 僵直率。濃度為 90.9 % 的電漿激活水對二齡幼蟲的 KT50 為 64 分鐘，且製備後放置室溫下 60 小時，仍然可對其造成 100 % 僵直率。在 90.9 % 和 98 % 濃度的電漿激活水的綠豆水耕植袋試驗的實驗結果顯示，不論預防組還是治療組，電漿激活水的處理對綠豆的生長情形皆無影響。意外的是，與對照組相比，澆灌電漿激活水的處理組中，根瘤線蟲的根瘤指數較嚴重且根瘤數量、卵塊數和後代數量較多。最後，以高濃度 98 % 電漿激活水進行小白菜的介質穴盤試驗，對根瘤線蟲的防治效果明顯，根瘤指數在對照組最高為 3，而處理組最低為 1；處理組的根瘤數量平均每株 1 ~ 2 個，低於對照組 3 ~ 7 個，且無任何卵塊和卵的產生。此外，不論在預防組或是治療組中，澆灌 98 % 電漿激活水的小白菜生長非常良好，在葉片面積、地上部高度和地上部鮮乾重均明顯大於對照組且達顯著差異。 本論文為首篇探討電漿激活水對植物寄生線蟲的防治效力報告，結果顯示電漿激活水是具有應用潛力之非化學藥劑防治資材，且兼具促進小白菜生長之功效。未來在農業產業鏈中，可將電漿激活水的運用，加入根瘤線蟲病害的綜合管理策略中。

Root-knot nematodes ( Meloidogyne sp. ) are the plant parasitic nematodes of glabal economic importance with broad host range. They induce rootknots on host roots that affect the development of roots, cause poor growth, leaf yellowing, and stunting and lead to poor quality and quantity of crops. For many years, farmers mainly rely on nematicides to control the root-knot nematodes. However, most of the nematicides contain neurotoxins that would heavily jeopardize human health and badly impact the environment. Recent years, several nematicides with high toxicity have been banned due to the raising awareness of sustainable agriculture. However, many of the remained applicable nematicides share the same mode of actions, which likely have gradually led to nematicidal resistance and ineffective management in the fields. Therefore, there is an urgent need for new a nematode control method. Plasma is the fourth state of matter, ionized gas in nature. Plasma activated water ( PAW ) can be produced by diffusing plasma into water. Based on the anti-microbial characteristic of plasma in previous stuidies, this research aimed to investigate the feasibility of applying PAW for root-knot nematode management. This research used the root-knot nematodes collected from Xizhou township of Changhua county as a target, and the Dielectric Barrier Discharge ( DBD )-prepared PAW as a testing material. With multiple dilute concentration, the in vivo direct killing ability, the in situ pre- and post- infection nematode control efficacy in pot experiments were evaluated. First, the nematode species were identified as Meloidogyne enterolobii and Meloidogyne incognita by the perineal pattern, PCR and gene sequence alignment. Then, 100% plasma activated water was produced by a system that use air to push plasma into ddH2O, and has the characteristics of pH 3, electric conductivity about 300μs/cm, and oxidation reduction potential of 580mv. After soaking the 2nd stage root-knot nematode juveniles in 75% diluted PAW for 2 hours, 100% of the nematode were expressing stiffness. The KT50 of PAW at a concentration of 90.9% for the 2nd stage juveniles was 64 minutes; Even if the PAW was placed at room temperature for 60 hours after preparation, it still reached the 100% nematode stifiness effect. The mung bean hydroponic bag inoculation experiments with treatments of 90.9% and 98% PAW showed that the PAW had no effect to plant growth; no matter if it was treated before or after the nematode inoculation. Unexpectedly, compare to the control group, the PAW-treated groups showed higher galling index and more galls, eggmasses and progenies. On Chinese cabbage, the nematode control with 98% PAW treatment was obvious. The maximum galling index of the control group was 3, and minimum was 1 for of the treatment group. Comparing the average of 3 to 7 galls per plant in the control group, the average number of galls in the treatment group was only 1 to 2 per plant, and without any eggmass. In addition, the Chinese cabbage treated with the 98% PAW grew very well; The leaf size, height, aboveground fresh and dry weights were all significantly larger than the control group. In sum, this is the first efficacy investigation report of the PAW application for root-knot nematodes management. The result of this study showed that the not only PAW is a material with application potential as a non-chemical nematode management option, it also promotes the growth of Chinese cabbage. In the future, PAW application could be incoperated into the Integrated pest management ( IPM ) strategy against the root-knot nematode in the agriculture industrial chain.