西瓜甜瓜種子上細菌性果斑病菌之偵測

Abstract

本研究探討西瓜和甜瓜種子上瓜類細菌性果斑病菌(Acidovorax avenae subsp. citrulli)之檢測。為了提高檢測效率，種子上的病原菌首先以含有25ppm cefoperazone、10ppm ceftriaxone及25pp piperacillin三種抗生素的改良型半選擇性培養基WFB68增量後，比較ELISA、直接PCR及immunomagnetic separation — PCR (IMS-PCR)等方法的偵測效果。直接PCR法在以100粒人工污染種子(含1及5%帶菌率)為樣品的檢測上均呈正反應。但以200粒種子(含0.5及2.5%帶菌率)為樣品時，只有50%的驗出成功率。以1000粒種子(含0.1及0.5%帶菌率)為樣品時，西瓜種子利用ELISA及直接PCR均無法被偵測出，而IMS-PCR法則有80%的檢出率；甜瓜種子利用ELISA法不能被檢測出，直接PCR法對0.5及0.1%帶菌種子分別有40及20%檢出率，而IMS-PCR法分別有100及80%檢測成功率。檢測三批天然污染種子(兩批西瓜及一批甜瓜種子)，IMS-PCR法的偵測成功率(80―100%)亦較直接PCR法(22.5―62.5%)及ELISA法(0―40%)為高。防治藥劑處理種子不影響IMS-PCR在種子上之檢測。PCR反應所用的引子對SL1/SR1對國外果斑病菌供試的九株菌株亦具專一性，而另一引子對L2/R2只能增幅出屬於group II 菌株的預期產物。本研究結果顯示IMS-PCR在西瓜及甜瓜種子中果斑病菌的檢測上，具實際應用的潛力。

The objective of this study was to investigate the detection of Acidovorax avenae subsp. citrulli (Aac) in watermelon and melon seeds. For increasing the efficiency of detection, the infested seeds were incubated in an improved semiselective medium WFB68 containing 25 ppm cefoperazone, 10 ppm ceftriaxane and 25 ppm piperacillin. After enrichment with this medium, enzyme-linked immunosorbent assay(ELISA), direct polymerase chain rection (direct PCR) and immunomagnetic separation and polymerase chain rection(IMS-PCR) were compared for their efficiencies of detection. Detection by direct PCR for artificially infested seed samples(1 and 5% infestation) using 100 seeds as an assay sample were all positive, however, only 50% samples were positive using 200 seeds(0.5 and 2.5% infestation)as an assay sample. With 1000 seeds as an assay sample, Aac on watermelon seed samples (0.1 and 0.5% infestation) were not detected by ELISA and direct PCR, but 80% detection rate (positive tests per total tests) were obtained by IMS-PCR; Aac on melon seed samples(0.1 and 0.5% infestation) was also not detected by ELISA, but 20 and 40% detection rates were obtained by direct PCR, and 80 and 100% detection rates were obtained by IMS-PCR for seed samples with 0.1 and 0.5% infestation, respectively. When three naturally infested seed lots ( two of watermelon and one of melon seeds) were assayed, the detection rates by IMS-PCR(80 ―100% ) were higher than those by direct PCR(22.5 — 62.5%) and ELISA (0 ― 40%). Detection by IMS-PCR was not affected by seed treament with agrochemicals. The primer pair SL1/SR1 used in PCR was aslo specific for all nine strains of A. avenae subsp. citrulli from foreign countries, but the other primer pair L2/R2 only amplified a distinct DNA band from strains belonging to group II. The results indicate that the IMS-PCR could be potentially used as a seed-detection assay for A. avenae subsp. citrulli on watermelon and melon seeds.