Fig. 1. Nucleotide sequence alignments of ASSVd isolates and the position of ASSVd-specific primers and molecular beacon. Comparison of the nucleotide sequences of ASSVd isolate collected from ‘Hongro’ cultivar at the orchard of NIHHS with ASSVd-K (Korean strain, accession AF421195), A-4 (DQ362907), B-9 (DQ362906), AM1 (EU031455), A1 (HG764204), B1 (HG764201), C1 (HG764197), C2 (HG764198), C3 (HG764199), C4 (HG764200), T1 (HG764202), P1 (HG764203), N1 (HG764205). ASSVd isolate which identified in this study had 100% sequence homology with ASSVd-K. Identical nucleic acid residues are shown in black, whereas dashes indicate gaps in the nucleotide sequences introduced to optimize alignment. The primers and molecular beacons sequences for ASSVd are indicated by red rectangles.
Fig. 2. A dendrogram which constructed from the sequences of ASSVd isolates; accessions K (AF421195), B-9 (DQ362906), A - 4 ( D Q 3 6 2 9 0 7 ), A M 1 ( E U 0 3 1 4 5 5 ), A M 2 ( E U 0 3 1 4 5 6 ), P P 1 ( E U 0 3 1 4 6 7 ), PE1 (EU031477), AP1 (EU031487), C1 (HG764197), C2 (HG764198), C3 (HG764199), C4 (HG764200), B1 (HG764201), T1 (HG764202), P1 (HG764203), A1 (HG764204), N1 (HG764205), ap (HQ840722), and ADFVd (NC_003463), CBLVd (M74065), and PBCVd (NC_001830) belong to the genus Apscaviroid. It was constructed by the maxium likelihood method based on Tamura-Nei model with 1,000 bootstraps.
Fig. 4. The standard curve of ASSVd RNA using real-time NASBA. (A) Real-time NASBA amplification plot based on TTP value versus fluorescence. (B) Relationship of TTP to ASSVd RNA copy number.
Fig. 5. Comparison of sensitivity to detect ASSVd RNA molecules by RT-PCR and real-time NASBA. (A) End-point detection of ASSVd by RT-PCR. (B) Relative fluorescence with a dilution series by real-time NASBA.
Fig. 3. Sensitivity of ASSVd real-time detection using molecular beacon to determine the optimization of the assay. Optimization of (A) the primers, (B) KCl and (C) ITP concentration with the highest fluorescent signal or the lowest time to positive (TTP) value.
Table 1. Sequences of the primers and molecular beacon used in RT-PCR and real-time NASBA assays for detecting ASSVd in apple trees
References
- Boom, R., Sol, C. J., Salimans, M. M., Jansen, C. L., Wertheimvan Dillen, P. M. and van der Noordaa, J. 1990. Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28:495-503. https://doi.org/10.1128/JCM.28.3.495-503.1990
- Compton, J. 1991. Nucleic acid sequence-based amplification. Nature 350:91-92. https://doi.org/10.1038/350091a0
- Flores, R., Randles, J. W., Bar-Joseph, M. and Diener, T. O. 1998. A proposed scheme for viroid classification and nomenclature. Arch. Virol. 143:623-629. https://doi.org/10.1007/s007050050318
- Hassan, M., Myrta, A. and Polak, J. 2006. Simultaneous detection and identification of four pome fruit viruses by one-tube pentaplex RT-PCR. J. Virol. Methods 133:124-129. https://doi.org/10.1016/j.jviromet.2005.11.002
-
Hibbitts, S., Rahman, A., John, R., Westmoreland, D. and Fox, J. D. 2003. Development and evaluation of Nuclisens
$^{(R)}$ basic kit NASBA for diagnosis of parainfluenza virus infection with 'end-point' and 'real-time' detection. J. Virol. Methods 108:145-155. https://doi.org/10.1016/S0166-0934(02)00268-9 - Kim, H. R., Lee, S. H., Lee, D. H., Kim, J. S. and Park, J. W. 2006. Transmission of Apple scar skin viroid by grafting using contaminated pruning equipment and planting infected seeds. Plant Pathol. J. 22:63-67. https://doi.org/10.5423/PPJ.2006.22.1.063
- Koganezawa, H., Yang, X., Zhu, S. F., Hashimoto, J. and Hadidi, A. 2003. Apple scar skin viroid in apple. In: Viroids, eds. by A. Hadidi, R. Flores, J. W. Randles and J. S. Semancik, pp. 137-141. CSIRO Publishing, Collingwood, Australia.
- Kumar, S., Stecher, G., Li, M., Knyaz, C. and Tamura, K. 2018. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35:1547-1549. https://doi.org/10.1093/molbev/msy096
- Kwon, M. J., Hwang, S. L., Lee, S. J., Lee, D. H. and Lee, J. Y. 2002. Detection and distribution of apple scar skin viroid-Korean strain (ASSVd-K) from apples cultivated in Korea. Plant Pathol. J. 18:342-344. https://doi.org/10.5423/PPJ.2002.18.6.342
- Lee, J. H., Park, J. K., Lee, D. H., Uhm, J. Y., Ghim, S. Y. and Lee, J. Y. 2001. Occurrence of apple scar skin viroid-Korean strain (ASSVd-K) in apples cultivated in Korea. Plant Pathol. J. 17:300-304.
- Lee, S. H., Kim, Y. H. and Ahn, J. Y. 2016. The detection and diagnosis methods of infectious viroids caused plant diseases. J. Life Sci. 26:620-631. https://doi.org/10.5352/JLS.2016.26.5.620
- Lee, S. H., Ahn, G., Kim, M. S., Jeong, O. C., Lee, J. H., Kwon, H. G., Kim, Y. H. and Ahn, J. Y. 2018. Poly-adenine-coupled LAMP barcoding to detect Apple scar skin viroid. ACS Comb. Sci. 20:472-481. https://doi.org/10.1021/acscombsci.8b00022
- Liu, F. C., Chen, R. F. and Chen, Y. X. 1957. Apple scar skin disease. Academia Sinica Printery, Beijing, China. 43 pp (in Chinese).
- Menzel, W., Zahn, V. and Maiss, E. 2003. Multiplex RT-PCRELISA compared with bioassay for the detection of four apple viruses. J. Virol. Methods 110:153-157. https://doi.org/10.1016/S0166-0934(03)00112-5
- Nakahara, K., Hataya, T. and Uyeda, I. 1998. Inosine 5'-triphosphate can dramatically increase the yield of NASBA products targeting GC-rich and intramolecular base-paired viroid RNA. Nucleic Acids Res. 26:1854-1855. https://doi.org/10.1093/nar/26.7.1854
- Nemeth, M. 1986. Virus, mycoplasma, and rickettsia diseases of fruit trees. Springer Netherlands, Dordrecht, The Netherlands. 750 pp.
- Shamloul, A. M., Faggioli, F., Keith, J. M. and Hadidi, A. 2002. A novel multiplex RT-PCR probe capture hybridization (RTPCR- ELISA) for simultaneous detection of six viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid, and Pospiviroid. J. Virol. Methods 105:115-121. https://doi.org/10.1016/S0166-0934(02)00090-3
- Ushirozawa, K., Tojo, Y., Takemae, S. and Sekiguchi, A. 1968. Studies on apple scar skin disease. 1. On transmission experiments. Bull. Nagano Hort. Exp. Res. Stn. 7:1-12 (in Japanese).
- Vaskova, D., Spak, J., Klerks, M. M., Schoen, C. D., Thompson, J. R. and Jelkmann, W. 2004. Real-time NASBA for detection of strawberry vein banding virus. Eur. J. Plant Pathol. 110:213-221. https://doi.org/10.1023/B:EJPP.0000015378.27255.12
- Yang, X., Hadidi, A. and Hammond, R. W. 1992. Nucleotide sequence of apple scar skin viroid reverse transcribed in host extracts and amplified by the polymerase chain reaction. Acta Hortic. 309:305-309. https://doi.org/10.17660/actahortic.1992.309.44
- Zuker, M. and Stiegler, P. 1981. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 9:133-148. https://doi.org/10.1093/nar/9.1.133