水稻白尖病與徒長病之快速檢測技術建立及應用

Abstract

水稻之種子傳播性病害如水稻白尖病及水稻徒長病嚴重影響稻米產量。由於造成兩病之病原，水稻葉芽線蟲(Aphelenchoides besseyi)和水稻徒長病菌(Fusarium fujikuroi)皆能存於穀粒中，並於育苗時期感染水稻苗，若能於水稻育苗時期進行種苗篩選，則能有效管理此二病害於田間之發生。因此，開發快速分子檢測技術應用於育苗階段，對於健康種苗流程的建立至關重要。本研究利用恆溫圈環形核酸增幅法 (Loop-mediated isothermal amplification, LAMP) 建立兩病害之快速檢測技術，將其最佳化並應用於水稻種苗檢測。針對水稻葉芽線蟲之mtDNA COI與rDNA ITS 區塊共設計5組LAMP引子組，挑選其中增幅效率最佳之引子組AB-ID37，在反應溫度63oC下進行敏感性和專一性測試。敏感性結果顯示，反應時間為90分鐘可偵測出的最低濃度為103 copies/µl，且亦可於60分鐘內偵測出單隻線蟲抽取之核酸。專一性測試的結果，也證實無須擔心其他地上部植食性線蟲可能會造成之偽陽性反應。另外，在水稻苗上之應用性測試，則確認其偵測的敏感性可達到每株苗5隻。在水稻徒長病菌的偵測技術開發方面，針對其cps/ks gene共設計3組LAMP引子組。挑選增幅效率最佳之引子組CPS-ID4，在反應溫度63oC下進行敏感性和專一性測試。敏感性結果顯示，當目標核酸濃度為50 pg/μl以上時可在90分鐘的反應時間內被成功偵測。專一性測試的結果亦顯示本試驗技術可有效排除Fusarium屬中其他相近種之檢出。以人工接種之水稻苗進行應用性測試，顯示該技術可以成功偵測已受到濃度104 spores/ml感染但尚未發病之水稻苗。將此兩組LAMP引子組與前人開發之專一性PCR引子對比較，在敏感性方面雖無顯著優勢，但專一性遠高於傳統PCR檢測。將本研究中開發之LAMP引子組，搭配hydroxy naphthol blue (HNB) 和lateral flow strip之應用，皆可於偵測終端快速顯示偵測結果，有效減少完成檢測判讀所需時間，亦大幅提升本技術應用於水稻苗期檢測和植物檢疫的潛力。

Rice white tip disease caused by the leaf and bud nematode (Aphelenchoides besseyi) and the Bakanae disease caused by the fungus Fusarium fujikuroi are both seed-borne pathogen that result in huge yield-loss on rice worldwide. A rapid pathogen screening method that could be applied on symptomless rice seedling would effectively lower the diseases incidence in rice fields. The purpose of this study was to develop a loop-mediated isothermal amplification (LAMP) method for A. besseyi and F. fujikuroi detection. For the nematode detection, five sets of primers targeting COI gene region of mitochondial DNA or the ITS region of ribosome gene of A. besseyi were designed and the most efficient set named AB-ID37 was chosen for assay optimization. The AB-ID37 LAMP assay is capable of completing within 90 minutes with the detection sensitivity as low as 103 copies/µl of cloned plasmid DNA. The crude DNA of single nematode can be detected in 60 minutes with the assay. The AB-ID37 LAMP assay is with high specificity and is concern-free for false detection from amplifying other aboveground plant-parasitic nematodes. On the other hand, three primer sets targeting the cps/ks gene of F. fujikuroi were designed and the most efficient set, CPS-ID4, was chosen for Bakanae disease detection optimization. The CPS-ID4 LAMP assay is capable of completing within 90 minutes at the 50 pg/μl DNA detection sensitivity. The CPS-ID4 LAMP assay can distinguish F. fujikuroi from other closely related Fusarium spp. when tested with whole genomic DNA. Inoculums of several concentrations of nematodes and fungi on single rice seedling were also examined and the assay was shown to be effective with no false positive or negative results. Although these two LAMP assays did not show better sensitivity when comparing with other published PCR detection methods, they are more spieces-specific and suit better for detection and screening purposes. When hydroxy naphthol blue (HNB) and lateral flow strip were applied along with the LAMP reaction, the instant visual and result analysis shorten the overall assay completion time and highly increased the potentiality of this assay in field and quarantine applications.