電漿激活水對根瘤線蟲發育之調控

Abstract

象耳豆根瘤線蟲 (Meloidogyne enterolobii) 為新興植物病原，其寄主廣泛且具有強致病力，可突破已知作物中對其他根瘤線蟲之抗性，對番茄、番石榴等重要經濟作物危害甚鉅。田間一般以化學藥劑防治根瘤線蟲，但其長久累積易造成環境負擔，其非專一性危及非目標生物，且對施用者存在較高健康風險。電漿激活水 (Plasma-activated water, PAW) 為電漿化後的大氣與水反應之產物，為新型環境友善資材。過往研究指出，PAW對象耳豆根瘤線蟲二齡幼蟲具有致死效果，且能顯著抑制相關病徵發展與線蟲繁殖，使植株生長較好，為具有應用潛力的線蟲病害防治資材。本研究從線蟲生理與轉錄體層面探討PAW於象耳豆根瘤線蟲之作用機制，期望強化該資材未來於植物線蟲病害防治之應用基礎。本研究發現，浸泡於PAW中的卵發育速度變慢甚至停滯；有74%無法順利發育成一齡幼蟲，其餘26%發育至一齡期後即停止生長。於PAW中，卵孵化的速度較慢，且孵化率顯著降低70%。二齡幼蟲接觸PAW後6小時，死亡率為35%；而若接觸24小時，則死亡率則可達83%。以細胞滲透螢光探針H2DCFDA檢測二齡幼蟲體內活性氧物質(Reactive oxygen species, ROS)總量，發現經過PAW處理24小時的組別含量增加35%，暗示PAW在短時間內對線蟲造成氧化壓力而影響其生存。透過接種試驗發現，二齡幼蟲在PAW環境中對番茄之侵染率下降65.3%。於番茄盆栽實驗中澆灌PAW，在接種後第7、21和35天皆觀察到發現已侵入根內的線蟲發育速度仍然緩慢，第35天僅25%能發育至成蟲。在接種後第35天，根部的根瘤數量相較未處理組降低40%。同時，觀察到經過PAW處理後的族群子代中不僅雄蟲比例上升，族群規模亦下降。在盆栽試驗中亦觀察到澆灌電漿水可有效抑制番茄病徵發展使植株生長情況較佳。本研究利用RNA定序技術 (RNA-sequencing) 探討象耳豆根瘤線蟲二齡幼蟲接觸電漿激活水後轉錄體之變化。分析結果獲得75個差異表現基因 (differentially expressed genes, DEGs)。其中，25個基因表現量上升，50個基因表現量下降。透過與COGs、GO和KEGG資料庫比對，表現量上升的DEGs主要參與細胞代謝與訊息傳導 (cellular processes and signaling)、定位(localization)、酵素活性(enzyme activity)、花生四烯酸代謝(arachidonic acid metabolism)與紫質代謝(porphyrin metabolism)。而表現量下降的DEGs主要參與細胞代謝(cellular processes)細胞凋亡(apoptosis)和離子結合(ion binding)。本研究結果闡明PAW對象耳豆根瘤線蟲生活史中自卵至成蟲的各階段皆有抑制作用，可能作用機制為影響根瘤線蟲細胞活性與脂質代謝而造成線蟲氧化壓力。

The guava root-knot nematode, Meloidogyne enterolobii, is an emerging plant pathogen with a wide host range and worldwide distribution. It is an aggressive parasite with high pathogenicity, which overcomes known resistance genes in plants and reproduce successfully on them. M. enterolobii causes enormous losses on important economic crops such as tomato and guava. Nematicide application is the most common and effective method for root-knot nematode management in the fields. However, long-term nematicide application damages the environment, harms non-target organisms, and poses high health risks to growers. Plasma-activated water (PAW) is a new eco-friendly material generated by treating water with cold atmospheric plasma. Previous study showed that PAW had lethal effect on the second-stage juveniles (J2) of M. enterolobii. In addition, PAW significantly reduced nematode disease symptoms on plants, inhibited nematode reproduction, and induced plant growth. Based on its high application potential in root-knot nematode management, this study aims to further investigate the physiological influences and unveil the genetic mechanisms of the regulation of M. enterolobii by PAW, in the hope of providing a solid foundation for PAW application on plant nematode diseases. In this study, PAW was found to inhibit the embryogenesis of M. enterolobii. 74% of the PAW-treated eggs stopped development before reaching the first-stage juveniles (J1), and the other 26% couldn’t grow beyond J1. Eighty-three percent J2s died after 24 hours after PAW treatment. The egg hatch was delayed by PAW, and the hatching rate decreased 70%. The total amount of reactive oxygen species (ROS) detected by H2DCFDA in the PAW-treated J2 increased by 35%. The result indicated that PAW caused oxidative stress on J2s in a short period and influenced their survival. Through inoculation plate experiment, the number of J2 successfully invading the tomato roots was decreased 65.3% in the PAW environment. When tomato plants were irrigated with PAW, the juvenile development was slowed down on the 7th, 21st, and 35th days post inoculation (dpi). At 35 dpi, only 25% became adults, and the number of root knots decreased 40%. At the same time, PAW changed the nematode population structure and size. The male ratio increased, and the number of offspring decreased. Meanwhile, PAW reduced nematode disease symptoms and promoted tomato growth. In addition, transcriptome analysis was performed to investigate the influence of PAW on the J2s. A total of 75 differentially expressed genes (DEGs) were identified. Among them, 25 genes were up-regulated and 50 genes were down-regulated. Through the annotations of the DEGs with COGs, GO and KEGG databases, the up-regulated genes were found to be enriched in cellular processes and signaling, localization, enzyme activity, arachidonic acid metabolism, and porphyrin metabolism, while the genes in cellular processes, binding and apoptosis were down-regulated. The study demonstrated that PAW had adverse effects on every development stage of M. enterolobii life cycle, from egg to adult, and that PAW also improves plant growth in pots. In addition, the results revealed that PAW caused oxidative stress to nematodes and regulates the cell activity and lipid metabolism of M. enterolobii.