開發臺灣草莓灰黴病帶菌果實之檢測方法

Development of PCR-based methods for rapid detection of Botrytis cinerea on strawberry in Taiwan

由Botrytis cinerea所引起之草莓灰黴病對草莓產量影響甚劇，此病原菌可感染花、果實或葉片等部位，由於此病原菌寄主廣泛、潛伏期長，經常在作物開花期感染後處於潛伏階段，直至果實成熟時或採收後始出現病徵，難以即時防治，導致作物產量嚴重損失。因此本研究擬利用一般聚合酶連鎖反應技術（conventional polymerase chain reaction, conventional PCR）及SYBR green螢光染劑即時聚合酶連鎖反應技術（SYBR green-based real-time PCR）來開發臺灣草莓灰黴病菌的帶菌果實檢測方法，建構出快速、省工的草莓帶菌果實之快速檢測流程，以及早診斷病害。文獻指出，草莓灰黴病菌專一性引子對C729F/C729R可搭配conventional PCR技術，檢測出草莓灰黴病菌；另一組引子Rpb2aF/Rpb2aR，可檢測出天竺葵上的灰黴病菌，惟該兩組引子對臺灣草莓灰黴病菌菌株之專一性尚未被探究，且臺灣對草莓灰黴病菌帶菌果實之分子檢測方法亦尚未被標準化。因此本研究擬先驗證引子對C729F/C729R及Rpb2aF/Rpb2aR對臺灣草莓灰黴病菌之專一性，之後搭配於conventional PCR、SYBR green-based real-time PCR之分子檢測技術與自動化核酸萃取方法，藉此建構出快速、省工的草莓帶菌果實快速檢測流程。結果顯示：比較兩組引子於conventional PCR及SYBR green-based real-time PCR技術中之檢測靈敏度，Rpb2aF/Rpb2aR對檢體（包含Standard DNA、Genomic DNA、Conidia）的靈敏度約十倍於C729F/C729R。依兩組引子搭配自動化核酸萃取流程所建立之分子檢測方法，於SYBR green-based real-time PCR系統中，皆可檢測出不同罹病程度的草莓果實（包含未出現病徵之帶菌果實），兩組引子對帶J. Plant Med.Vol. 65 No. 3, 2023 103DOI:10.6716/JPM.202309_65（3）.0003菌檢體之檢出率皆為100%，惟依據引子組Rpb2aF/Rpb2aR所開發之檢測方法，對檢體的靈敏度較高，因此優先推薦依據該引子對搭配於SYBR green-based real-time PCR與自動化核酸萃取方法之分子檢測流程，做為臺灣草莓灰黴病菌帶菌果實之標準化分子檢測方法。此檢測方法可應用於草莓採收後之分子檢測上，希望未來於病害發生前即能檢測出病原，藉此擬定預防或控制病害的適當策略減少因病原快速傳播而造成的草莓果實供應商之損失，同時達到監控田間降低糧食損失之目的。

Gray mold caused by Botrytis cinerea has a severe impact on strawberry yield. This pathogen can infect various parts of the plant, including flowers, fruits, and leaves. Due to the broad host range and long latent period, infection often occurs during the flowering and immature fruit period, with symptoms appearing only after harvest. As a result, it is challenging to prevent and control the disease, leading to significant crop yield losses. Therefore, this study aimed to develop a rapid and labor-saving detection method for B. cinerea in strawberries using conventional polymerase chain reaction (PCR) and SYBR green-based real-time PCR techniques for Taiwanese farmers. This method can facilitate the rapid detection of asymptomatic infected fruits, providing an early diagnosis of the disease. According to literatures, the specific primers for B. cinerea, C729F/C729R, can be used in conjunction with conventional PCR to detect the pathogen, and another primer set, Rpb2aF/Rpb2aR, can detect gray mold pathogens on geraniums. However, the specificity of these two primer sets for B. cinerea strains in Taiwan has not been investigated, and a standardized molecular detection method for B. cinerea-infected strawberry fruit in Taiwan has not been established. Therefore, this study aims to verify the specificity of the primer sets C729F/C729R and Rpb2aF/Rpb2aR for B. cinerea strains in Taiwan, and then combine them with molecular detection techniques such as conventional PCR and SYBR green-based real-time PCR, as well as automated nucleic acid extraction methods, to construct a rapid and efficient detection process for B. cinerea-infected strawberry fruit. The results showed that the sensitivity of Rpb2aF/Rpb2aR in detecting samples (including standard DNA, genomic DNA, and conidia) was approximately ten times higher than that of C729F/C729R in both conventional PCR and SYBR green-based real-time PCR. Using the molecular detection methods established by combining the two primer sets with an automated nucleic acid extraction process, different levels of diseased strawberry fruits (including symptomless carrier fruits) could be detected in the SYBR green-based real-time PCR system. The detection rates of both primer sets for carrier samples were 100%, but the detection sensitivity of the method developed based on the Rpb2aF/Rpb2aR primer set was higher. Therefore, it is recommended to prioritize this primer set for the standardized molecular detection of Taiwan's strawberry gray mold carrier fruits using the SYBR green-based real-time PCR and automated nucleic acid extraction methods. This detection method can be applied to molecular detection after harvested strawberry. We hope to detect the pathogen before the occurrence of the disease in the future, formulate appropriate strategies for disease prevention or control, and reduce losses to strawberry fruit suppliers caused by the rapid spread of pathogens.