Abstract:Regions representing about 80% of the 16S rDNA sequences of nine phytoplasma isolates were amplified by polymerase chain reaction (PCR). These partial 16S rDNA sequences amplified from the various phytoplasmas were used in DNA heteroduplex mobility assays (HMA). Based on DNA distances derived from HMA analysis, the nine phytoplasma isolates may be classified into four distinct groups: group I, paulownia witches'-broom (China), potato purple top (France); group II, tomato stolbur (France); group III, eastern aster yellows (U. S.A. ), faba bean phyllody (Sudan), vinca phyllody (Sudan), tomato big bud (Australia); group IV, crotalaria witches'-broom (Thailand), clover proliferation (Canada).

Abstract:DNA amplification by polymerase chain reaction (PCR) was employed to detect the mycoplasma-like organism (MLO) associated with paulownia witches'-broom (PaWB). A 1.2 kb DNA fragment of PaWB MLO was amplified with primer pair R16F2R2. A minimum of only 16pg total nucleic acids from infected paulownia tissue culture maintained at 25-28°C was needed to detect PaWB MLO. An appropriate concentration of total nucleic acids for amplification used for detection of PaWB MLO from the tissue culture was demonstrated to be 240 pg/,ul to 6.5 ng/,ul. PaWB MLO DNA content in total nucleic acids from MLO-infected tissue culture was as high as 5 times that from MLO-infected paulownia plant grown at 23-25°C in the greenhouse. Results showed that the amplification by PCR is highly effective in detecting the low concentrations of PaWB MLO known to occur in irregular patterns in diseased paulownia plants.

Abstract:This study focused on the purification and characterization of chitinase from alfalfa (Medicago sativa L.) infected by CP-MLO. Chitinase (EC 3.2.114) was purified from leaves and shoots of MLO-infected alfalfa plants by ammonium sulfate precipitation, chitin affinity chromatography and Sephadex column chromatography. Three chitinase isoenzymes were identified and shown to have molecular masses of approximately 40, 35 and 30 kilodaltons as determined by SDS-polyacrylamide gel electrophoresis, while only one chitinase isoenzyme was detected in healthy plants. The endochitinase activity in CP-MLO infected plants was 24 times higher than that in healthy plants. The optimum temperature range of the enzyme activity was from 35°C to 50°C. Ag and Hg ions significantly inhibited the enzyme activity.

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