草莓根腐線蟲之鑑定與偵測技術開發及其田間族群動態之研究

根腐線蟲 (Pratylenchus spp.) 寄生超過300種植物，在全球重要作物造成嚴重經濟損失。本實驗室2017年於苗栗大湖地區之草莓園GS及KDL發現大量根腐線蟲，對其進行型態量測及分子鑑定，確認物種為 P. vulnus (草莓根腐線蟲)，為台灣新紀錄種。本研究以接種試驗確認其病原性後，為提供快速準確之鑑定及監測方法，開發專一性的分子即時定量檢測技術。此外，對此種根腐線蟲於草莓園中的族群消長進行超過一年的監測，透過分析生物性與非生物性因子，探討與田間病原性真菌之交互作用可能性，並建立適用田間之族群預測模型。本研究中發現，大湖草莓園所分離得之族群型態量測平均值符合該物種最早被報導的California族群數據範圍，但與最新被報導的Lorca Chiha族群，則有雄成蟲數據差異。四個分子標記區域進行基因親緣性分析的結果，均確認物種分類地位與型態測量結果，且暗示本族群已特化獨立於其他地區族群。接種試驗結果顯示P. vulnus確實能在草莓根部繁殖並造成明顯之壞疽病徵，但對植株的生長勢並無影響。運用粒線體COI基因作為為標的所開發的專一性PCR引子對PvC2978F / PvC2978R，經測試及優化後，無論以土壤或根部組織的全核酸樣本進行試驗，均能成功進行目標偵測。以其作為基礎，後搭配專一性探針PvC-pr的使用，亦開發即時定量的PCR偵測技術。此偵測技術的準確性，經130個樣本的檢測定量分析與改良式柏門氏漏斗分離法相比，顯示兩方法所得的數量雖有正相關性，但本方法偵測所得結果最多可多15540倍，凸顯本qPCR技術之準確性。在田間族群動態分析方面，非生物性因子中僅有每月土壤平均溫度與每月根腐線蟲密度有顯著負相關性。族群密度會隨著草莓植株種植天數 (Day after planting, DAP) 增加而明顯上升，且DAP與每月土壤平均溫度有負相關性，並呈現二項式分佈。以DAP:112作為分組切線，成功建立兩組在不同DAP情況下，能以每月土壤平均溫度預測P. vulnus田間族群密度之模型。此外，以本研究另外開發之專一性qPCR技術，對田區樣本中草莓尖鐮孢菌(Fusarium oxysporum f. sp. fragariae)進行定量，發現該真菌病原與之P. vulnus族群密度有正相關性，暗示交互作用的存在。然而，雙重病原的接種交互作用的溫室接種試驗中，於草莓生長勢的分析，數據結果僅顯示草莓尖鐮孢菌為主要效應。

關鍵字

草莓根腐線蟲；分子檢測技術；族群動態；預測模型；草莓尖鐮孢菌

並列摘要

Pratylenchus spp. parasitize more than 300 plant species globally and cause damages and enormous economic loss on important crops. In 2017, large amount of Pratylenchus spp. nematodes were discovered in two strawberry fields in the Dahu area of Miaoli county. Both morphological measurement and molecular identification results indicated the nematode species as Pratylenchus vulnus; a new record species in Taiwan. Inoculation of the nematode on strawberry had confirmed its pathogenicity. To establish an accurate and quick method for diagnosis and monitoring, a specific quantitative real-time PCR method was developed. Next, the population dynamic of P. vulnus in the strawberry field was monitored for over a year and the correlations between biotic and abiotic factors and the nematode density was studied. Finally, a population density prediction model was built and the interaction between the nematode and pathogenic fungi was analyzed.This study found that the body measurement means of P. vulnus Taiwan isolate fall in the same range of the California isolate. On the other hand, the data from male body measurements are somehow different to the Lorca Chiha isolate. The California isolate and the Lorca Chiha isolate are the earliest and the latest P. vulnus group being reported. sequence analysis of four molecular markers not only confirmed the morphological identification, but also further implied the possibility of an isolated evolution of P. vulnus Taiwan isolate. The inoculation of P. vulnus on strawberry confirmed its capability of causing significant lesion symptoms on the root but does not influence the plant growth. For the detection assay development, a specific PCR primer set PvC2978F / PvC2978R was designed to target the COI gene region of mtDNA. Optimization results showed the successful detection of the target nematode in total DNA of root tissue and soil. With an addition of the specific PvC-pr probe, the quantitative real-time PCR assay was established. The qPCR quantification results of 130 soil samples indicated the positive correlation to the results obtained by Baermann funnel method. However, the data gap is up to 15540 fold-time. From the population dynamic analysis, only the average monthly soil temperature was significantly negatively correlated to the monthly nematode density. The population density significantly was found to increase along with DAP (day after planting). The DAP is negatively correlated to the average monthly soil temperature in binomial distribution. Therefore, when separate the data using DAP:112, two population density prediction models were established; Both models use the monthly soil temperature for P. vulnus density prediction, but are for applications in difference DAP ranges. In addition, with the specific qPCR assay developed in this study, the amount of the strawberry pathogenetic fungus Fusarium oxysporum f. sp. fragariae was quantified in the soil samples. The result showed a positive correlation of the fungus and the population density of P. vulnus. However, the result of interaction experiment only revealed the impact of the fungus on the strawberry growth.