日日春葉片黃化病之病原植物菌質體與其媒介昆蟲之探討

Abstract

於2005年8月起，桃園縣大園鄉之園藝花卉栽培區陸續發現疑似植物菌質體 (phytoplasma) 感染所造成之日日春病害。依罹病植株所呈現之葉片黃化、簇葉、花器葉化等病徵，將此ㄧ新興病害命名為日日春葉片黃化病 (periwinkle leaf yellowing, PLY)。初步利用聚合酵素連鎖反應 (polymerase chain reaction, PCR) 檢測出罹病植株中帶有植物菌質體之基因序列，因此本論文進一步針對該植物菌質體之核醣體RNA基因序列 (rDNA) 進行分析，發現罹病植株體內之植物菌質體乃屬於第一群 (16SrI group) 植物菌質體（又稱為翠菊黃萎病群 (aster yellows group, AY group) 植物菌質體），並且經由嫁接實驗與電顯切片觀察後，進一步確認該病原菌之存在情形，故將其命名為日日春葉片黃化病植物菌質體 (periwinkle leaf yellowing phytoplasma, PLY phytoplasma)。研究中針對PLY phytoplasma所屬之第一群植物菌質體與其他各群植物菌質體之rDNA序列進行比對，設計出第一群植物菌質體專一性之PCR 引子對，以供後續田間罹病植株檢測以及媒介昆蟲之調查。本研究由2007年起，進行媒介昆蟲帶菌情形與族群數量變化之月份監測，同時也將檢測出帶菌之媒介昆蟲進行分類鑑定，至今已發現當地有四種葉蟬可攜帶PLY phytoplasma，分別為東方二叉葉蟬 (Macrosteles orientalis Vilbaste)、二點透翅角頂葉蟬 (Cicadulina bipunctella)、黑點角頂葉蟬 (Phlogotettix cyclops) 以及二室葉蟬 (Balclutha sp.)。2007年5月至2009年4月間之檢測結果顯示，當地媒介昆蟲可於三月至十月間測得帶菌情形，而每年最早測得帶菌之媒介昆蟲皆為東方二叉葉蟬，其帶菌月份恰與當地日日春之栽種時間（約在每年三月下旬至九月底）大致吻合，且其族群數量亦於這段期間內大幅提升。於2008年夏季，採集帶菌率較高之東方二叉葉蟬與二點透翅角頂葉蟬，針對溫室栽培之健康日日春進行傳菌實驗，結果證實此二種葉蟬確實具有傳播PLY phytoplasma至日日春之能力。本研究中也以同步聚合酵素連鎖反應 (real-time PCR) 進行罹病日日春以及四種帶菌蟲體體內帶菌量之測定，在測定帶菌量時，同時藉由測定DNA模板中寄主（日日春或葉蟬）DNA質量 (ng) 以作為校正基準之方式，可更準確的比較感染後不同時間點之罹病日日春以及四種帶菌葉蟬體內帶菌量之差異。由定量之結果顯示，每單位重量 (ng) 之東方二叉葉蟬DNA模板帶菌量非常高，甚至高於嚴重發病之日日春。整合上述研究成果，當地發現之四種可帶菌葉蟬中，東方二叉葉蟬在日日春葉片黃化病之發病生態中扮演較重要之角色，而其他三種葉蟬則可能屬於次要之傳播媒介。

In 2005, periwinkle plants exhibiting symptoms of yellowing, witches’ broom, phyllody and virescence were observed in flower production fields in Dayuan, Taoyuan county. The sequences of 16S rDNA and 16S-23S rDNA intergenic spacer region of the causative agent of this newly discovered periwinkle leaf yellowing (PLY) disease were amplified with polymerase chain reaction (PCR) using the total DNA prepared from the diseased leaves as a template. Sequence analysis of 16S rDNA revealed that the causative agent of PLY was closely related to the phytoplasmas of the aster yellows group (16SrI group) those cause worldwide diseases in many horticultural and vegetable crops. The results of grafting experiment and TEM observation provided further evidences to support that the phytoplasma is the causative agent of PLY. The periwinkle leaf yellowing phytoplasma was thus named “Candidatus Phytoplasma asteris” strain PLY. According to the rDNA sequences of various 16SrI group phytoplasmas, the PCR primer pair AYTWf1/ AYTWr1 specific for 16SrI group phytoplasma was designed to detect the PLY phytoplasma in the plants and insect vectors. At present, four species of PLY phytoplasma-harbored leafhoppers were captured in Dayuan and identified as Macrosteles orientalis Vilbaste, Cicadulina bipunctella, Phlogotettix cyclops and Balclutha sp., respectively. Monthly PCR detection indicated that insect vectors harbored PLY phytoplasma from March to October in the year of 2008, and Macrosteles orientalis Vilbaste was the earliest PLY phytoplasma-carrier detected during the period of 2007 to 2009. Transmission experiments with field-collected Macrosteles orientalis Vilbaste and Cicadulina bipunctella showed that both leafhoppers can transmit the PLY phytoplasma to healthy periwinkle plants. The titer of PLY phytoplasma in diseased periwinkle plants and four species of PLY phytoplasma-harbored leafhoppers were quantified in relation to plant or insect DNA (genome units [GU] of phytoplasma DNA per nanogram of plant or insect DNA) using a quantitative real-time PCR. It was also proved that Macrosteles orientalis Vilbaste contains the highest titer of PLY phytoplasma than the other leafhoppers. Therefore, it is reasonable to conclude that Macrosteles orientalis Vilbaste plays a major role in the epidemiology of PLY disease, and the importance of the other leafhoppers call for further investigation.