蒲瓜種子上附著的胡瓜綠斑嵌紋病毒之檢測

Abstract

利用實驗室先前保存的胡瓜綠斑嵌紋病毒（CGMMV）本地胡瓜分離株及其抗血清進行本試驗。以汁液磨擦接種，本株病毒可感染南瓜以外的供試葫蘆科植物，但不感染任何供試番茄及茄子品種。綠藜及圓葉菸草可被系統性感染，因此用來當繁殖寄主。蒲瓜帶CGMMV的種子洗出液當檢體，經系列稀釋，比較5種病毒檢測方式，結果能有效檢出的檢體稀釋終點分別為：1（生物分析）、10^(-3)（酶聯抗體免疫分析）、10^(-3)（免疫電子顯微鏡）、10^(-2)（西方墨漬法）、10^(-4)（反轉錄聚合酶連鎖反應）。收集蒲瓜‘強力1號’，種子3個批號（seed lots）進行種子檢查，先以群體測試方式準備樣本，再以間接法酶聯抗體免疫分析檢測，最後以“seedhealth ”程式估算各批種子中CGMMV檢出率及信心範圍，分別是0%（1877T-1212）、0.93±0.64%（1877T-2B）及82.71±10.17%（9881V-AA）。以批號9881V-AA的種子播種進行出芽試驗，有74.8%的苗株種皮對CGMMV抗體呈正反應，僅有0.5%的苗株子葉呈正反應，本葉則全數呈負反應。因此確認CGMMV感染蒲瓜時僅污染其中的種皮，並未侵染至種皮以內的胚或胚乳，因CGMMV病毒顆粒很穩定，檢測種皮或其種子洗出液即可檢出病毒。綜合以上結果，得以解釋CGMMV雖然種子帶原（seed-carried）的比率很高，但是能經種子垂直感染子代苗的比率卻很低。 A domestic cucumber isolate of Cucumber green mottle mosaic virus (CGMMV) and from which the homogenous antiserum raised previously were applied in this study. By mechanical inoculation, this virus could infect all of the tested cucurbitaceous plants except Cucurbita spp., but none cultivars of tomato or eggplant were infected. Chenopodium murale and Nicotiana benthamiana were systemically infected and therefore were used as CGMMV propagating hosts. Serially diluted suspensions which eluted from CGMMV-carried bottle gourd seeds were comparatively detected by 5 methods. The final dilution end points of effective detection were: 1 (infectivity), 10^(-3)0 (ELISA), 10^(-3) (IEM), 10^(-2) (western blot), and 10^(-4) (RT-PCR), respectively. Three seed lots of bottle gourd (Lagenaria siceraria) cv. 'S-1' were examined for seed health testing. Tested seeds of each seed lot were prepared for group testing and all specimens were detected by indirect ELISA. By computer program "seedhealth," the proportion of CGMMV infected seeds in each seed lot and the confidence limit for 95% confidence level were estimated as 0% (1877T-1212), 0.93±0.64% (1877T-2B) and 82.71±10.17% (9881V-AA). Seeds from CGMMV contaminated seed lot 9881V-AA were adopted for 'growing on' test. Even 74.8% specimens collected from testa were positively detected by ELISA, the CGMMV positive proportion was only 0.5% of those from cotyledons, and 0% from primary leaves. It means that most of the testa-existed CGMMV do not distribute to the embryo or endosperm of infected seeds. However, due to the stability of virion, CGMMV was readily detected in specimens of testa or seed elute. Based on the results of this study, we reveal the fact that although the high proportion of CGMMV was carried by infected seeds, the incidence of vertical transmission of CGMMV infecting the progeny seedlings is very low.