• The Plant Pathology Journal
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• v.21 no.1
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• pp.55-58
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• 2005

In order to diagnose and differentiate jujube witches' broom (JWB) phytoplasma rapidly, oligonucleotide primer pair, 16Sr(V) F/R, for polymerase chain reactions (PCRs) was designed on the basis of 16S rRNA sequences of JWB phytoplasma. The PCR employing phytoplasma universal primer pair P1/P7 consistently amplified DNA in all tested phytoplasma isolates. But no phytoplasma DNA was detected from healthy jujube seedlings. The nested PCR, the primer pair 16S(V) F/R, about 460 bp fragment, amplified DNA in all tested JWB and related phytoplasmas including ligustrum witches' broom phytoplasma of the 16S rRNA group V, but no DNA amplification was detected from other phytoplasma strains such as groups 16SrI (Aster yellows) and 16SrXII (Stolbur group) in which mulberry dwarf phytoplasma and chrysanthemum witches' broom phytoplasma belong to, respectively. The same results were obtained from both Korean and Chinese isolates of JWB phytoplasma. Nested-PCR using phytoplasma universal primer pair P1/P7 and 16SrV group-specific primer pair 16S(V) F/R could detect group V phytoplasmas rapidly and easily, in particular JWB phytoplasma.

• Journal of Korean Society of Forest Science
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• v.96 no.6
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• pp.737-741
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• 2007

Typical phytoplasma witches' broom symptoms were observed in black locust (Robinia pseudoacacia L.) in Korea. The symptoms of the disease were showing abnormally small leaves, shortened intemodes and proliferation of shoots. The phytoplasmas were detected consistently in all the symptomatic samples by the amplification with phytoplasma universal primer pairs P1/P7 and R16F2n/R2, and the expected size was 1.8 kb and 1.2 kb. However, the phytoplasma DNA was not detected in healthy seedling. Based on sequence analysis of amplified region, this phytoplasma has close homologies with aster yellow, mulberry dwarf, maize bushy stunt, ash witches' broom and sumac witches' broom phytoplasmas, more than 99.2% but showed homologies with black locust witches' broom (GeneBank Accession No. AF 244363), and jujube witches' broom, 88.6% and 87.7%, respectively. This phylogetic analysis indicates that the black locust witches' broom phytoplasma founded in korea should be classified in the Candidatus phytoplasma asteris (16Sr I) group and clearly distinct from the black locust witches' broom group 16Sr III (peach X-disease phytoplasma group).

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.73.2-73
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• 2003

Typical whiches broom symptoms caused by phytoplasma were observed in Ash (Fraxinus rhynchophylla Hence) in Korea. The symptoms were showing abnormally small leaves, short internodes, and proliferation of shoots. Fluorescence and electron microscopy of leaf midribs revealed phytoplasma positive DAPI fluorescence and numerous phytoplasma bodies localized in the phloem sieve tubes. Phytoplasma DNA of 1.8 Kb was detected consistently from all symptomatic samples by the amplification of phytoplasma DNA with the phytoplasma specific primer pair Pl/P7. But no phytoplasma DNA was detected in healthy ash seedlings. Based on sequence analyses of an amplified region, this phytoplasma is closely related to Eqilodium phyllody, Mulberry dwarf, and Aster yellows phytoplasmas with the homology of 99.95 ％, 99.79 ％ and 99.78 ％, respectively, This phylogenetic analyses indicate that ash witches broom phytoplasma but is evidently distinct from the ash yellows group 16SrⅦ and should be classified into the Aster yellows group 16SrⅥ.

• Research in Plant Disease
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• v.17 no.3
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• pp.265-271
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• 2011

Seven phytoplasma diseases have been occurred on floricultural crops in Korea : Ph-ch1 and Ph-ch2 of chrysanthemum, Ph-lily of lily, petunia flat stem-Korean (PFS-K) of petunia, poinsettia branch inducing- Korean (PoiBI-K) of poinsettia, statis witches' broom-Korean (SWB-K) of statis and azalea witches broom (AWB). Classification of the seven phytoplasmal diseases based on 16S ribosomal RNA (rRNA) sequences showed that floricultural crop phytoplasma disease were widespread in order of aster yellow (AY), stolbur and X-disease in Korea. In phenotypic characters, the fasciation was occurred in both monocotyledon plant of lily and dicotyledon plants of petunia and poinsettia. Besides, the fascination was occurred in Ph-lily of stolbur, petunia PFS-K of AY and PoiBI-K of X-disease. This result indicated that phytoplasma classification based on 16S rRNA and symptoms are not consistently related. The comparison of 16S rRNA sequence of the seven floricultural crop phytoplasma with five tree phytoplasmal diseases of jujube witches' broom, paulownia witches' broom, wild jujube witches' broom, mulberry dwarf, golden rain phytoplasma occurred in Korea showed as high as 88.5-99.9% homology. Among them, especially mulberry dwarf showed the highest homology with the seven floricultural crop phytoplasms. Based on this result, floricultural crop phytoplasmas were assumed to be transmitted by insect vectors from tree phytoplasmas in Korea.

• The Plant Pathology Journal
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• v.29 no.4
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• pp.465-470
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• 2013

In January 2012, spreading type petunia cv. Wave Pink plants showing an abnormal growth habit of sprouting unusual multiple plantlets from the lateral buds were collected from a greenhouse in Gwacheon, Gyeonggi Province, Korea. The presence of phytoplasma was investigated using PCR with the primer pairs P1/P6, and R16F1/R1 for nested-PCR. In the nested PCR, 1,096 bp PCR products were obtained, and through sequencing 12 Pet-Stol isolates were identified. Comparison of the nucleotide sequences of 16S rRNA gene of the 12 Pet-Stol isolates with other phytoplasmas belonging to aster yellows or Stolbur showed that Pet-Stol isolates were members of Stolbur. The presence of phytoplasma in petunia was also confirmed by microscopic observation of the pathogens. In this study, Stolbur phytoplasma was identified from spreading type petunia cultivars by sequence analysis of 16S rRNA gene of phytoplasma and microscopic observation of phytoplasma bodies. This is the first report of Stolbur phytoplasma in commercial Petunia hybrida cultivars.

• Journal of Korean Society of Forest Science
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• v.101 no.4
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• pp.635-639
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• 2012

The present study was initiated to find out the insect vector of mulberry dwarf phytoplasma. Among the collected insects from mulberry cultivation region, the sucking insects (Hishimonas sellatus, Bothrogonia japonica and Balclutha punctata) were selected for transmission of mulberry dwarf phytoplasma. Sucking insects, H. sellatus and B. punctata successfully transmitted phytoplasma to healthy seedling mulberry and periwinkle plants by inoculation-feeding. Typical dwarf symptom was observed in mulberry tree, but very slender twigs on stem and formatted small leaves were found on periwinkle plants, which are typical witches' broom symptoms. The phytoplasmas were detected in all samples exception sucking insects B. japonica using phytoplasma universal primer pair R16F2n/R2.

• Journal of Korean Society of Forest Science
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• v.95 no.6
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• pp.631-635
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• 2006

Single-strand conformation polymorphism (SSCP) analysis of MD and JWB phytopalsma isolates which amplified PCR products using the R16F2n/R2 phytoplamsa universal primer pair were compared for variations of their nucleotide sequence. The MD and JWB phytoplasmas were clearly distinct each of the band patterns from about 1.2 kb PCR products. To clearly distinct of close SSCP band patterns, the MD and JWB phytoplasma PCR products were mixed and performed to detect their polymorphism. The SSCP band patterns show all of bands of MD and JWB on single lane and easily distinct their each band patterns. The PCR-SSCP analysis was possible to detect of 1.2 kb nucleotide sequence and near close band patterns were easily distinct by mixing two samples.

• Journal of Korean Society of Forest Science
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• v.100 no.4
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• pp.623-629
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• 2011

The detectable levels and population fluctuations of phytoplasmas infecting dwarf mulberry trees were investigated using nested-PCR and competitive-PCR methods. Samples of five different types were studied : A. petiole of a leaf that displays dwarf symptoms, B. petiole from apparently healthy leaf residing on a branch also supports a leaf with dwarf symptoms, C. the branch portion that supports a leaf with dwarf symptoms, D. the leaf petiole from healthy appearing leaves on branch with no dwarf symptoms, and branch portion of branch with no dwarf symptoms, E. the rootlets of trees with dwarf symptoms. These 5-parts were collected from each tree during June - April, once in every two months. The phytoplasma was detected from all parts of collected mulberry samples during all seasons using nested-PCR with AS-1/AS-2 primer pairs. The phytoplasma was detected until $10^4$ dilution using direct-PCR method, but it was detected until $10^{13}$ dilution by the nested-PCR method. The density of pytoplasma was found to be $7.94{\times}10^{18}-10^{12}copies/{\mu}L$ in mulberry trees. The density of phytoplasma was observed throughout the year in all samples of mulberry trees. The highest rates of phytoplasma was found in the samples B and C during the early growing season followed by the sample A and D during the dormant season. Samples C and E displayed the highest phytoplasma density followed sample D. The density of phytoplasma appeared stable during all the seasons for samples C and A. The result of the present study demonstrates the utility of nested-PCR and competitive-PCR for detection and determination of population fluctuations of phytoplasmas in plant tissues.

• Journal of Korean Society of Forest Science
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• v.102 no.2
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• pp.223-228
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• 2013

Phytoplasmas were detected consistently in 42 mulberry cultivars showing dwarf disease using DNA analysis by amplification with phytoplasma universal primer pairs P1/P7 (about 1.8 kb and R16F2n/R2 (about 1.2 kb). The point mutation from 42 cultivars of mulberry tree was detected by single-strand conformation polymorphism (SSCP) analysis. The SSCP profiles were clearly observed from all of cultivars in 8% polyacrylamide gel, electrophoresizing for and running 8-15 hrs. at 150V, $10^{\circ}C$. The MD and JWB phytoplasma PCR products was mixed and electrophoresis was performed to detect their polymorphism. In this results, the SSCP profiles of all bands of MD and JWB were analyzed on single lane and were distinct in their each of band patterns. The SSCP analysis was possible to detect of 1.8 kb and 1.2 kb nucleotide size and near close band patterns were distinct by mix of two samples. Previously, it was only possible to detect of point mutation under 600 bp nucleotide sequence by SSCP analysis but this modification of SSCP technique was possible to detect clearly SSCP band patterns of about 1.8 kb and 1.2 kb nucleotides.