以PCR與quantitative PCR技術開發水稻紋枯病之快速檢測法

Development of the rapid detection methods of rice sheath blight based on PCR and quantitative PCR techniques

沈世茂(Shen Shih-Mao)

指導教授 ： 林盈宏

國立屏東科技大學/農學院/植物醫學系所/碩士(2018年)

https://doi.org/10.6346/THE.NPUST.PM.003.2018.D02

摘要

水稻紋枯病菌 (Rhizoctonia solani) 會造成水稻紋枯病 (sheath blight of rice)。此病原菌為土傳性病原菌，能以菌核及菌絲殘存於土壤中，可自莖基部侵入水稻植株並於葉鞘部大量繁殖，進而造成水稻發生枯萎的虎斑狀病徵，為全球水稻產業上一大重要限制因子。為了預防此病害發生造成水稻產業上的經濟損失，發展一個具備專一、快速、且能夠現地進行檢測之診斷工具用以檢測田間之R. solani，就此病害管理而言是重要策略之一。本研究擬開發一個水稻紋枯病菌之現地檢測技術，用以檢測R. solani。根據試驗結果，本研究已開發出快速萃取試劑，可用於田間檢體之核酸萃取，並完成專一性引子對SMS RS1-F/ SMS RS1-R及GMRS-3/ITS1靈敏度及專一性測試。本研究以四種不同萃取系統進行比較，結果顯示自動萃取系統搭配專一性引子對SMS RS1-F/ SMS RS1-R之檢出率為100%，為四種萃取系統中最穩定；而使用快速萃取試劑次之，其檢出率為96%，此結果證實本研究所開發的快速萃取試劑確實能有效地用於檢測紋枯病。透過同日間分析 (intraday assay) 及異日間分析 (interday assay) 測試結果，證實本研究所開發的不同萃取系統皆能夠穩定地進行水稻檢體的核酸萃取。以不同病程之水稻檢體進行萃取之in-planta試驗結果顯示，以自動萃取系統進行檢體核酸萃取可獲得較穩定的檢出率，平均檢出率高於80%。未來擬進一步針對田間帶菌土壤進行萃取測試，使此檢測系統更能落實於水稻田間紋枯病菌之現地檢測工作中。

關鍵字

水稻紋枯病菌 ； 快速萃取試劑

並列摘要

Sheath blight of rice (ShB) caused by Rhizoctonia solani is a major limiting factor to rice production. This soil-borne fungal pathogen can survive in the soil by sclerotia and mycelia. They are the primary inoculum in rice fields. The primary inoculum has ability to infect the rice sheath from the stem base and multiply in the tissues causing lesion symptoms on sheath. In order to prevent the outbreak of rice sheath blight and to reduce the economic loss of rice yield, it’s critically important to develop a rapid detection method with specific, fast, and on-site advantages for disease management. In this study, the rapid extraction reagent which can be used for on-site detection was developed. The specificity and sensitivity of the novel primers set SMS RS1-F/SMS RS1-R and the reference-primer set GMRS-3/ITS1 were tested. In addition, this study developed the four different extraction protocols for rice sheath infected by R. solani. Moreover, the intraday- and interday-assays were performed for the reproducibility evaluation of the detection methods developed in this study. The results of reproducibility evaluation indicated that all developed detection protocols were suitable to be used for diseased rice sheath. In the future, an on-site will be developed for field soil to make the detection system to be more completely.

並列關鍵字

Rhizoctonia solani ； PCR ； SYBR green-based real-time PCR ； TaqMan probe-based real-time PCR

參考文獻

參考文獻

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