費洛蒙與環境因子對水稻葉芽線蟲的影響

Abstract

水稻葉芽線蟲Aphelenchoides besseyi所引起的種傳性病害——水稻白尖病能造成高達70%的減產，為生產水稻重要的限制因子。針對種傳性病害，一般推薦使用預防型的防治策略，而研究線蟲的行為與相關調控機制將有助於相關防治策略的擬定。前人研究已知線蟲的發育與行為皆與環境中非生物因子相關，且受ascaroside類的費洛蒙調控。有鑒於近年氣候變遷，水稻白尖病的發生趨於嚴重，本研究在建立水稻葉芽線蟲的齡期分離技術後，運用此技術探討該線蟲費洛蒙對吸引異性及取食偏好性的行為影響，以及該線蟲生長發育受到環境中溫度與二氧化碳濃度的可能影響。首先，本研究結合並改良了可大量收集卵的齡期同步化技術，以及可收集成蟲的蔗糖懸浮法，成功建立水稻葉芽線蟲的齡期分離技術。其次，運用粗萃取與HPLC-MS/MS技術，本研究發現水稻葉芽線蟲ascarosides的釋放跟生長階段相關；在混合齡期 (ascr#9) 及二齡幼蟲 (J2) (ascr#7，oscr#9) 的樣本中所偵測到的ascarosides種類不同。本研究以行兩性生殖的水稻葉芽線蟲GD分離群為材料進行費洛蒙趨化性實驗，然而結果顯示雌蟲和雄蟲對於單一齡期或性別的費洛蒙粗萃物的反應並不具專一性，推測水稻葉芽線蟲吸引異性的費洛蒙需要在特定情況下產生。此外，本研究證實了水稻葉芽線蟲對於不同的水稻品種具有取食偏好性，且ascarosides對於其取食偏好性的影響與品種具有高度相關性。以人工合成的ascarosides， asc#7進行趨化性實驗，結果發現水稻葉芽線蟲的J2及成蟲皆會排斥高濃度之asc#7，暗示著asc#7與線蟲族群密度的調控可能相關。此外，本研究發現，用於人工培養線蟲的真菌Alternaria citri也會產生ascarosides，且利用S-basal溶液能收集到較多種類。自溫度與二氧化碳濃度的試驗中，發現溫度升高至26°C和28°C會加快葉芽線蟲的卵孵化和發育；由試驗數據可推測26°C有可能為水稻葉芽線蟲所造成的經濟損失趨於嚴重的閥值溫度。然而，水稻葉芽線蟲的卵孵化及存活率皆不受升高至800 ppm的二氧化碳濃度影響。整體而言，本研究建立了水稻葉芽線蟲齡期分離技術，證實了水稻葉芽線蟲A. besseyi及真菌A. citri會產生ascarosides並提供了其作為族群溝通訊號調控葉芽線蟲種內及種間反應的可能性，亦了解氣候變遷環境條件對水稻葉芽線蟲發育影響。

The leaf and bud nematode Aphelenchoides besseyi is a major limiting factor of rice production as the highest yield loss due to the white tip disease caused by this seed-borne nematode up to 70%. Prevention strategy is usually applied for seed-borne disease, and the investigation of plant-parasitic nematode behavior and its regulation mechanisms would help to establish a management strategy. Previous studies have shown that the nematode development and behavior are greatly influenced by the environmental abiotic factors, and regulated by a class of allelochemical called ascarosides. Given the fact that the white tip disease became severe due to climate change, the A. besseyi stage separation technique was established for the examination of ascarosides effect on the nematode behavior, including sex attraction and feeding preference; and the possible effect of temperature and carbon dioxide on nematode development. First, the study had optimized and combined the synchronization and sucrose flotation methods, that was for mass collection of eggs and adults, respectively; and established a productive stage separation assay for A. besseyi successfully. Then, by using nematode crude allelochemicals and HPLC-MS/MS technique, the study revealed that the ascarosides expression in A. besseyi was dependent on the developmental stages as different types of ascarosides were detected in the mixed-stage (ascr#9) and second-stage juvenile (J2) samples (ascr#7, oscr#9). In our study, the chemotaxis assay was performed with crude allelochemicals from GD strain that was amphimixis; however, the results showed that the behavior response of female and male was not gender- or developmental stage-specific, indicating that the sex attraction-related ascarosides may only produce under specific circumstances. In addition, the feeding preferences of A. besseyi towards different rice cultivars was confirmed in our study; and the ascarosides effect on feeding preferences was also rice cultivar-dependent. The results from the chemotaxis assay performed with synthesis ascarosides ascr#7 showed that all J2 and adults were repelled to the high concentration asc#7, indicating the possibility of ascr#7 involvement in A. besseyi population density regulation. Besides, the fungus Alternaria citri that used for nematode culture also produced ascarosides, and more types of ascarosides were collected through S-basal-collect produce. The results from temperature and carbon dioxide experiments showed that both elevated temperature (26°C and 28°C) accelerated the egg hatching and development rate. Also, from the experiment results, 26°C may suggest as the temperature threshold of the economic loss caused by A. besseyi became severe. However, the egg hatching and survival rates remained unaffected under elevated carbon dioxide of 800 ppm. In conclusion, our study established A. besseyi stage separation assay, revealed that A. besseyi and its fungal host, A. citri produce ascarosides, and provided the possible role of ascarosides as a population signal to regulate the A. besseyi intra- and inter-specific interaction; finally determined the effect of climate change on A. besseyi development.