Amplification of Paulownia Phytoplasma DNA Fragments by Random Primed-PCR.

Abstract

Abstract:Polymerase chain reaction (PCR) was performed to detect genomic differences between healthy and paulownia witches'-broom (PaWB)-affected paulownia plants using fifteen primer-pair combinations derived from five randomly synthesized primers based on the P1-like adhesin gene sequence of Mycoplasma pneumoniae. Upon comparing the PCR product profiles between healthy and PaWB-affected paulownia DNA samples, three types of PCR profiles were observed. Firstly, some bands were amplified from both healthy and PaWB-affected paulownia DNA samples; secondly, some DNA bands were amplified only from PaWB-affected paulownia DNA samples; thirdly, a few DNA bands were amplified only from healthy paulownia DNA samples. DNA amplified from PaWB-affected paulownia DNA samples hybridized only with PaWB-affected paulownia DNA samples, suggesting the DNA was derived from paulownia phytoplasma genome.Key words: Diagnosis; phytoplasma genome; gene cloning.Introduction. Phytoplasmas are associated with diseases in many plant species (Sears and Kirkpatrick, 1994), but the inability thus far to isolate phytoplasmas in pure culture has made them become among the most difficult plant pathogens to diagnose rapidly and accurately. Traditionally, in phytoplasma studies, pathogen identification and classification have relied primarily on comparative studies and their biological properties such as symptoms induced in diseased hosts, identity of plant species susceptible to infection, and specificity of phytoplasma transmission by insect vectors (Schaff et al.,1992). The recent development of phytoplasma-specific molecular probes for DNA hybridization assays has provided a rapid and reliable means of phytoplasma diagnosis and classification (Hiruki, 1992;Lee et al. 1993a;Sears and Kirkpatrick, 1994). To date molecular differentiation of similar phytoplasma strains has been studied mainly on the basis of DNA sequence homologies and DNA polymorphisms (Kuske et al., 1991;Lee et al., 1991Lee et al., , 1993aLee et al., , 1994Namba et al., 1993;Schneider et al., 1993). Currently, two common schemes for classification are in use; one based on polymorphisms in 16S rDNA (James et at., 1993;Lee et al., 1993a;Namba et al., 1993;Zhong and Hiruki, 1994) and the other on homology of a few random fragments of chromosomal DNA (Kirkpatrick et at., 1987;Kuske et at., 1991;Lee et at., 1991Lee et at., , 1993bNakashima et at., 1992). To fully understand genetic relationships among a variety of phytoplasmas and their molecular genetic composition, the use of only a few DNA fragment sequences for differentiation of phytoplasmas is not sufficient. Therefore, there is an urgent need to clone a variety of DNA fragments from phytoplasma genomes. However, traditional phytoplasma DNA cloning methods involve several time-consuming and laborous procedures, such as random DNA cloning of total plant DNA extracted from plants infected with phytoplasma, and random DNA hybridization selection. Recently, Williams et al. (1990) developed a procedure by which polymorphic

Paulownia

“…The disease incidence rate of Paulownia at 5 years old was approximately 50-80%, which could reduce 25% of timber production. Primers based on the P1-like adhesion gene sequence of Mycoplasma pneumoniae have been successfully used to detect this disease (Zhong and Hiruki 1994). If PWB disease is found, the host seedlings should be immediately removed.…”