利用real-time PCR進行花生簇葉病菌質體質體pPNWB套組數之測定與台灣梨衰弱病菌質體之檢測

Abstract

A plasmid, named pPNWB, was isolated from peanut witches’ broom phytoplasma (PnWB phytoplasma) in our lab previously. To estimate the copy number of pPNWB in PnWB phytoplasma cell, absolute quantification was implemented by real-time PCR with previously reported single copy gene, rpoB, in PnWB phytoplasma as a reference sequence. Variation of the copy number of pPNWB was found in different PnWB phytoplasma-infected periwinkle plants in this study. The copy number of pPNWB in PnWB phytoplasma cell increased as the number of PnWB phytoplasma cell increased in periwinkle plant after grafting was noticed. Same real-time PCR-based absolute quantification strategy was also applied to determine the copy number of 16S rRNA gene (16S rDNA) in PnWB phytoplasma. The PnWB phytoplasma, a 16SrII phytoplasma, was revealed to harbor two copies of 16S rRNA gene in it’s genome. The result is the same as those of other phytoplasma members of 16SrI, 16SrIII and 16SrX group. PDTWII phytoplasma, one of the causative agent of PDTW disease, is also the member of group 16SrII and may also carry two copies of 16S rDNA in it’s genome. The copy number of 16S rDNA, which is relatively easier to be cloned from phytoplasma than other genes, can be used as a reference sequence to quantify the number of phytoplasm cell in plant tissue. Pear decline in Taiwan (PDTW) was first reported in 1994 in orchards of central Taiwan. PDTW phytoplasma can be transmitted by two pear psyllas, Cacopsylla qianli and C. chinensis. PDTWII phytoplasma was first identified in pear psyllas in 2005. In 2006, the co-infection of PDTW phytoplasma and PDTWII phytoplasma was also evidenced in pear trees. To detect these two phytoplasmas in pear plants and insect vectors, the specific primer pairs for multiplex real-time PCR and real-time PCR were designed and applied effectively in this study. Many DNA samples prepared from PDTW phytoplasma-infected pears confirmed by PCR previously were thus reexamined using real-time PCR. PDTWII phytoplasma was detected in many of these samples. The results indicated that the co-infection of PDTW and PDTWII phytoplasmas in pear trees and inscet vectors may be very common in fields. The multiplex real-time PCR and real-time PCR-based detection also showed higher sensitivity than those of multiplex PCR and conventional PCR. The real-time PCR-based quantification of PDTW and PDTWII phytoplasmas established in this study will be applied to monitor the fluctuations of these two phytoplasmas’ populations in pear trees and insect vectors.