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The phylogenetic relationships of 11 phytopathogenic
phytoplasmas (or mycoplasmalike organisms) established on the
basis of restriciton fragment length polymorphisms(RFLP) of 16S
ribosmal RNA genes and sequences analyses of 16S-23S rDNA
spacer regions. Approximately full length of 16S rDNAs of 11
phytoplasma strains were amplified by polymerase chain reaction(
PCR) using the method develooped by Schneider et al.. The
procedure, depending on the presence of a BclI restriction site
in the 16S rDNA of chloroplasts but not in that of
phytoplasmas, permits the specific amplification of the 1.5 kb
16S rDNA of phytoplasmas from BclI-digested total DNA from
infected plants rsing primer pair fD1' and rP1' from conserved
regions of this gene. A total of 21 restriction enzymes was
used in RFLP analyses of the 1.5 kb phytoplasma 16S rDNA
fragments. Among these restriction enzymes, Alw44I, AocI,
NheI, PstI, XbaI and XhoI did not digest the 1.5 kb phytoplasma
16S rDNA fragment. Based on the phylogenetic tree developed
from RFLP data, these 11 phytoplasmas could be classified into
4 groups. The other part of this study, the spacer regions
between the 16S rRNA gene and 23S rRNA gene in the phytoplasma
rrn operon were amplified by PCR usint two primer pairs. Only
one spacer fragment was amplified in each phytoplasma except
loofah witches' broom(LWB) phytoplasma. There were three
amplified spacer fragments (LWB-1, LWB-2 and LWB-3) in LWB
phytoplasma. The amplified DNA was sequenced, but the
sequencing of paulownia witches' broom phytoplasma spacer was
not completed. The phytoplasma 16S-23S spacer region contains
a single tRNA (for Ile); however, threr is no tRNA in the
spacer of LWB-1 and LWB-3. In multiple alignmetn of
phytoplasma spacers, only tRNA and two short segments, which
may be involved in rRNA processing, appears to be conserved.
The phylogenetic tree constructed by the sequence data
suggested these 10 phytoplasmas to be classified into 4 groups.
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