萊豆(Phaseolus limensis)種子傳染菜豆普通嵌紋病毒(BCMV)與胡瓜嵌紋病毒(CMV)的特性

Abstract

2008年農戶自留及市售之萊豆種子播種後幼苗發生皺葉嵌紋的比率達96％，疑似病毒感染種子所致。目前台灣萊豆發生的病毒主要是菜豆普通嵌紋病毒（Bean common mosaic virus, BCMV）及胡瓜嵌紋病毒（Cucumber mosaic virus, CMV）。比較生物分析、酶聯抗體免疫分析（ELISA）、免疫點漬法、及反轉錄聚合酶連鎖反應（RT-PCR）等四種病毒檢定法，在病葉汁液中，BCMV的稀釋終點均為10^(-3)；CMV以RT-PCR檢測的稀釋終點為10^(-6)，其他檢測的稀釋終點都為10^(-4)。以直接組織轉漬檢測病株莢果，可見BCMV與CMV都分佈於莢、珠柄與種皮，但未分佈於子葉。莢果內部組織以ELISA檢測，也能確定病毒相同的局限分佈情形。利用接種感染BCMV或CMV的萊豆植株所生產的種子進行出芽試驗（growing-on test），其苗株都無病毒及病徵發生。罹病種子採收一年後，取樣1015顆種子中，已無BCMV或CMV被ELISA檢出者。採集8位農戶自留種，測得種子帶BCMV與CMV的比率分別為12.5％和41.3％，另7個來源市售種子，帶病毒率則分別為3.8％和4.8％。以市售種子進行出芽試驗，在540棵苗株中測得BCMV與CMV的感染率均為0.19％。根據結果顯示萊豆植株感染BCMV或CMV後，病毒均會聚集於莢組織，經由珠柄入侵種子，待種子成熟乾燥後，兩種病毒都僅存在於種皮，且存活率隨貯藏期降低，因此BCMV或CMV經種子傳染（seed-transmitted）子代苗的比率均低於種子帶病毒（seed-carried）的比率。 Lima bean (Phaseolus limensis) seedlings with symptoms of wrinkled and mosaic pattern on leaves were found on about 96% of tested grown-out seeds obtained from growers and market shops of various locations. The currently prevalent viruses of lima bean found in Taiwan are Bean common mosaic virus (BCMV) and Cucumber mosaic virus (CMV). The virus indexing methods of bioassay, enzyme-linked antibody immunoassay (ELISA), immuno-dot blotting, and reverse transcription-polymerase chain reaction (RT-PCR) were applied to compare their sensitivity on detecting BCMV and CMV from infected tissues and seeds of lima bean. We have found that BCMV could be effectively detected by all four aforementioned methods from crude sap preparations of infected lima bean leaves at a dilution end-point (DEP) of 10^(-3) while CMV could be detected at the DEP of 10^(-4) by biological, ELISA and immuno-dot blotting analyses and DEP of 10^(-6) by RT-PCR. Direct tissue blotting (DTB) was applied to detect the virus distribution in the diseased seed pod. Both BCMV and CMV could be detected at pod, funicle, and testa, but not at cotyledon. ELISA tests have confirmed the viruses distributed in internal tissues of the seed pods as the same as the results obtained by DTB. No symptoms and viruses have been observed on and detected in all tested seeds harvested from BCMV- or CMV- inoculated lima bean plants in growing-on tests. After a storage duration of one year, both BCMV and CMV were undetectable in the seeds of inoculated lima bean plants (1015 seeds in total) by ELISA. The ratios of seed-borne viruses in lima bean seeds were examined by ELISA. The results showed that there were 12.5% and 41.3% of grower’s seeds were infected by BCMV and CMV, respectively, while 3.8% and 4.8% of commercialized seeds were infected by BCMV and CMV, respectively. The incidences of seed-borne BCMV and CMV were both determined as 0.19% in a total of 540 seedlings derived from commercial seeds and indexed by grow-on tests. Accordingly, during the infection of BCMV or CMV to lima bean plants, viruses accumulate in the tissues of pod first, then invade seeds through the funicle. After maturation and dehydration of seeds, the BCMV and CMV exist only in the testa and their survival rates reduce as the storage duration extend. This, therefore, can be a possible explanation for the ratio of seed-transmission is lower than that of seed-borne of BCMV or CMV.