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This study determined the tuf gene sequence of the phytoplasma specific to paulownia witches’-broom from Nanyang, China (hereby designated PaWB-Ny). The PaWB-Ny tuf gene was 1185 nucleotides in length and confirmed that the phytoplasma belongs to subgroup 16SrI-D of aster yellows. Three characteristic GTP-binding protein motifs were identified based on the peptide deduced from the tuf gene sequence. Results suggested that the elongation factor EF-Tu was localized in the cytoplasm and lacked hydrophobic transmembrane domains. Antibodies against PaWB-Ny EF-Tu were prepared by rabbit immunization with glutathione-S-transferase (GST)-tagged EF-Tu fusion protein expressed in Escherichia coli. EF-Tu exhibited a molecular weight of ∼43 kDa and was detected in PaWB-infected paulownia plants by western blot analysis. Indirect enzyme-linked immunosorbent assays (ELISA) and dot blotting analyses were performed with freezing and thawing treatments during antigen preparation. Dilution of extracts to an appropriate scale significantly reduced non-specific reactions. The resultant PaWB EF-Tu antibody reacted with antigens from plants infected with periwinkle virescence and chinaberry tree witches’-broom phytoplasmas, but not those infected with jujube witches’-broom or bishopwood witches’-broom phytoplasma. The EF-Tu was characteristically localized within the phytoplasmal cytoplasm of infected plant phloem tissues. [ABSTRACT FROM AUTHOR]