鳳梨釋迦衰弱病的病原鑑定及落花生簇葉病分子偵測技術改良

植物菌質體 (phytoplasmas)係為一群韌皮部侷限的植物寄生性細菌，最早由日本數名科學家Doi等人於西元1967年率先發現，當時認為該病原體為mycoplasma-like organisms (MLOs)。至西元2020年為止，植物菌質體依照16S rDNA序列共分為34個大群 (groups)，其中落花生簇葉病屬於第二大群 (16SrII)的植物菌質體。目前的研究顯示，植物菌質體傳播的方式是經由刺吸式口器的昆蟲，如二點小綠葉蟬、南斑葉蟬等。受植物菌質體感染的寄主植物會出現以下幾種病徵：小葉化、花器葉化、簇葉、矮化、葉片黃化、巨芽等。本研究將進行探討的項目為不同引子對 (primers)對落花生簇葉病之聚合酶連鎖反應 (Polymerase Chain Reaction, PCR)檢測的敏感度差異及近年來疑似由植物菌質體感染的病害——鳳梨釋迦衰弱病——之病原鑑定。本研究的結果顯示，使用本實驗設計的引子對進行聚合酶連鎖反應 (Polymerase Chain Reaction, PCR)比起以往的廣效型引子對P1/P7擁有更清晰、明顯的條帶 (band)，並且以10的倍數進行序列稀釋後，再分別進行PCR反應，結果顯示10000倍稀釋擁有最大的差異度，即使用本實驗設計的引子對Phyto1622 (暫定名)可與傳統廣效型引子對P1/P7產生最大的清晰度及敏感度差異。根據PCR反應及序列定序的結果，鳳梨釋迦衰弱病的病原係為第二大群 (16SrII)的植物菌質體，且與引起落花生簇葉病的植物菌質體有99 %的序列相似度，未來將確定傳播途徑是否與落花生簇葉病有關，以期進行更有效的病害管理及防治。

關鍵字

鳳梨釋迦衰弱病；落花生簇葉病；擬菌質體；植物菌質體；聚合酶連鎖反應

並列摘要

Phytoplasmas are phloem-limited plant pathogenic bacteria, which was found the earliest by japanese scientists, including Doi et al., in 1967. They were first considered to be mycoplasma-like organisms (MLOs). In 2004, phytoplasmas were given an official name by International organization of mycoplasmology, which is known as Candidatus Phytoplasma. By 2020, phytoplasmas are classified into 34 groups, according to the sequences of 16S rDNA. Present studies suggested that phytoplasmas are transmitted by sap-feeding insect vectors of the Order Hemiptera, take Amrasca biguttula and Orosius orientalis Matsumura for examples. Several symptoms can be clearly observed when plants are infected by phytoplasmas, inclusive of decline, yellows, witches’ broom, virescence, phyllody, proliferation and stolbur. This study aims to precisely detect an unknown pathogen which causes Custard Apple Decline and to compare the sensitivity of two universal primers P1/P7 and Phyto1622 (tentative name) through Polymerase Chain Reaction, (PCR). The result in this study reveals that the detection of phytoplasmas by Phyto1622 appears to show more visible and apparent bands. Additionally, ten-fold dilutions of nucleic acid, representing 100 to 104, detected by using P1/P7 and Phyto1622 shows the most dramatic differences in both visibility and sensitivity. In accordance with the detection consequences, pathogen resulting in Custard Apple Decline seems to belong to 16SrII Candidatus Phytoplasma. Besides, it shares 99% of sequence indentity with Candidatus Phytoplasma aurantifolia (16SrII-A). The main way of transmission of Candidatus Phytoplasma aurantifolia (16SrII-A) to custard apples still remains to be verified, which links to the efficiencies of disease control.