利用溫度處理與直接組織轉漬法增進夜來香種球病毒檢定準確性之研究

The improvement for the detection of tuberose mild mosaic potyvirus in tuberose bulbs by temperature treatment and direct tissue blotting

陳金枝(C. C. Chen) ； 張清安(C. A. Chang)

《植物病理學會刊》 8卷2期 (1999/06) Pp. 83-88

https://doi.org/10.6649/PPB.199906_8(2).0006

摘要

本省栽培之夜來香普遍受夜來香微嵌紋病毒(tuberose mild mosaic potyvirus, 簡稱TMMV) 之感染，感病植株葉片及花軸均會出現輕微嵌紋病徵。本實驗比較酵素連結抗體吸附法(ELISA) 與直接組織轉漬法(Direct tissue blotting, DTB) 對偵測夜來香組織中TMMV之效果，試驗發現當樣品中病毒濃度高時，DTB與ELISA檢測所得之病毒檢出率並無差異。但是當樣品中病毒濃度較低時，葉片樣品以ELISA檢測所得之病毒檢出率較DTB之結果高，此現象應與E L I S A結果可以數據化而較容易判斷有關；不過當檢定對象為種球時，結果顯示縱使病毒濃度低於ELISA檢出範圍，DTB法仍可獲得較高之病毒檢出率，此現象可能與病毒在種球頂端鱗葉中傾向於累積在特定位置，而容易以D T B法判別出來有關。總之，我們認為當檢測對象為夜來香葉片時，應以ELISA進行檢測，而檢測對象為夜來香種球時，則以DTB為較佳之選擇。由於夜來香種球採收後於下一季栽培前通常儲藏於5℃環境下，本實驗長期監測冷藏種球組織內所含TMMV之濃度變化，發現病毒濃度在冷藏一個月後約下降40％，二個月後即降低至ELISA所能檢測之臨界點範圍，冷藏六個月後則僅剩三分之一之種球仍可被ELISA檢測到病毒存在。不過如果應用DTB檢測法，則本試驗所有之冷藏處理種球均可被檢出病毒存在，此結果再度印證DTB之敏感度確實比ELISA高。本實驗進一步發現利用25℃回溫處理經長期冷藏之種球，可於二天內迅速提升種球組織內之病毒濃度，促使其達到百分之百被ELISA檢出之濃度範圍，同時也對DTB結果之判斷有正面之幫助。綜合本研究結果我們建議當檢定夜來香種球時，以DTB法配合25℃種球回溫處理二天，可增進將病毒檢出之效果，加上DTB在取樣操作上比ELISA更為簡便及有效率，極適合作為未來無病毒夜來香種球生產或進出口種球檢疫認證時，針對大量種球之標準檢定方法。

關鍵字

夜來香 ； 夜來香微嵌紋病毒 ； 溫度處理 ； 病毒檢定 ； 直接組織轉漬法

並列摘要

Tuberose mild mosaic potyvirus (TMMV) is widely distributed in tuberose (Polianthes tubero s a. L.) varieties in Taiwan, causing mild mosaic symptoms on the leaves and flower stems. In this study two serological techniques, i.e. enzyme-linked immunosorbent assay (ELISA) and direct tissue blotting (DTB), were compared for their sensitivity in the detection of TMMV from tuberose leaves and bulbs. ELISA was more sensitive than DTB in indexing tuberose leaves, as evidenced from the results that 94 out of 100 leaf samples were found as positive by the former, while only 89 samples were detected by the latter. However, when the tuberose bulbs were indexed, the sensitivity of DTB was considerably higher than that of ELISA. The former positively detected all of the infected bulbs, while the latter only detected 81% of the bulb samples. The possibility for the higher sensitivity to detect infected bulbs by DTB than ELISA was discussed. Based on tuberose grower's usual practice, bulbs served as planting material should be stored at 5 ℃ as soon as they were selected. However, concentration of TMMV in bulb tissues was found quickly decreased. After 6 months, only 33% of the infected bulbs could be successfully detected by ELISA as compared to 100% detection before storage. However, all the stored bulbs in this experiment were detected as positive by DTB. When the stored bulbs were removed from 5℃ and incubated at 25℃ for 2 days, TMMV in the bulb tissue enhanced to a concentration level readily detectable by ELISA. Based on these results, we recommend that DTB can be used in combination with 25℃ enhancement treatment as a model protocol for the routine indexing of tuberose bulbs. This protocol should also be useful in the plant quarantine process required when virus-free tuberose bulbs are to produce or export.

並列關鍵字

tuberose ； tuberose mild mosaic potyvirus ； temperature treatment ； virus indexing ； direct tissue blotting