Quadruplex PCR detection of three quarantine Phytophthora pathogens of berries
- 54 Downloads
In China, Phytophthora rubi, P. fragariae and P. × cambivora are quarantine organisms affecting berries for which rapid, specific and high-throughput methods are required for their simultaneous molecular detection. In this study, universal primers 18SUF/18SUR for the tested 13 Phytophthora species based on 18S rRNA sequences, specific primers PRSF/PRSR and PFSF/PFSR respectively for P. rubi and P. fragariae based on nad9 gene sequences, and specific primers PCSF/PCSR for P. × cambivora based on Ypt1 gene sequences were designed. The specificity of primers was demonstrated using 17 strains belonging to 13 Phytophthora species. A quadruplex PCR that specifically and simultaneously detects the three quarantine pathogens was developed. Application of this system detected an 884 bp universal band of 18S rRNA among all the Phytophthora species, a specific band of 556 bp and 615 bp to nad9 gene respectively in P. rubi and P. fragariae, and a 314 bp band specific to Ypt1 gene in P. × cambivora. Over conventional PCR, the sensitivity of the quadruplex PCR for P. fragariae was unchanged and lowered for P. rubi and P. × cambivora; but the specificity and throughput of the quadruplex PCR were all increased. This study represents the first molecular differentiation of P. rubi and P. fragariae, the two very similar Phytophthora species affecting berries. The quadruplex PCR developed can be applied for the rapid, specific and simultaneous detection in culture of the three quarantine Phytophthora species affecting berries and lays an excellent basis for further research to test it directly on plant materials and /or soil samples.
KeywordsPhytophthora fragariae Phytophthora rubi Phytophthora × cambivora Rubus spp. Simultaneous molecular detection Sensitivity tests
This work was funded by Rapid Identification of Quarantine Fungal Phytophthora Pathogens of Imported Fruits and Seedlings, a Programme from The General Administration of Quality Supervision, Inspection and Quarantine of PRC (2012IK286) and Plant Quarantine Pathogen Detection and Application by DNA Barcoding, National “the 12th Five-Year” Science and Technology Support Program (2012BAK11B02).
Compliance with ethical standards
This research did not involve any animal and/or human participants. The authors declare that they have no conflict of interests.
- Anderson, H. W. (1935). Black stele root rot of strawberry. Phytopathology, 25, 5.Google Scholar
- Duncan, J. M., Fordyce, W., Harper, P. C., & Rankin, P. A. (1986). Eliminating red core (Phytophthora fragariae) from Scottish certified stock strawberries. Research and Development in Agriculture, 3, 43–46.Google Scholar
- EPPO (2018) EPPO Global Database (available online). https://gd.eppo.int/taxon/PHYTFR/hosts.
- Félix-Gastélum, R., & Mircetich, S. M. (2005). Influence of flooding duration on the development root and crown rot of Lovell peach Prunus persica (L) Bstsch caused by three different Phytophthora species. Revista Mexicana de Fitopatología, 23, 33–41.Google Scholar
- Hickman, C. J. (1940). The red core root disease of the strawberry caused by Phytophthora fragariae n. sp. Journal of Pomology, 18(2), 89–118.Google Scholar
- Jung, T., Jung, M. H., Scanu, B., Seress, D., Kovács, G. M., Maia, C., Pérez-Sierra, A., Chang, T.-T., Chandelier, A., Heungens, A., van Poucke, K., Abad-Campos, P., Léon, M., Cacciola, S. O., & Bakonyi, J. (2017). Six new Phytophthora species from ITS clade 7a including two sexually functional heterothallic hybrid species detected in natural ecosystems in Taiwan. Persoonia, 38, 100–135.CrossRefGoogle Scholar
- Lacourt, I., Bonants, P. J. M., Van Gent-Pelzer, M. P., Cooke, D. E. L., Hagenaarde Wddrdt, M., Surplus, L., & Duncan, J. M. (1997). The use of nested primers in the polymerase chain reaction for the detection of Phytophthora fragariae and P. cactorum in strawberry. Acta Horticulturae, 439, 829–838.CrossRefGoogle Scholar
- Liu, Z. Y., Kang, Y. P., & Yuan, B. (2008). Identification of Phytophthora fragariae. Plant Protection, 34(5), 163–165.Google Scholar
- Liu, Y. T., Zhu, L. H., Li, P. J., Liao, F., Ren, X. Y., & Li, G. R. (2015). Triplex-PCR molecular detection of two quarantine fungal diseases of Prunus-Phytophthora syringae and P. cambivora. Journal of Plant Protection, 42(4), 571–577.Google Scholar
- McKeen, W. E. (1958). Red stele root disease of the loganberry and strawberry caused by Phytophthora fragariae. Phytopathology, 48, 129–132.Google Scholar
- Pepin, H. S. (1967). Susceptibility of members of the Rosaceae to races of Phytophthora fragariae. Phytopathology, 57, 782–784.Google Scholar
- Wang, H., Zhu, R. L., Tan, Y. L., Wei, K., Wang, X. J., Sun, Z. H., & Sheng, P. C. (2011). Establishment and application of multiple PCR for diagnosing Proteus mirabilis, Salmonella and Listeria monocytogenes. Scientia Agricultura Sinica, 44(11), 2334–2340.Google Scholar
- Werres, S. (1988). Enzyme-linked immunosorbent assay (ELISA) as a method for detection of Phytophthora fragariae in strawberry roots. Nachrichtenblatt des Deutschen Pflanzenschut- zdienstes, 40, 146–150.Google Scholar
- Wilcox, W. F. (1997). Phytophthora root rot. In M. A. Ellis, R. H. Converse, R. N. Williams, & B. Williamson (Eds.), Compendium of raspberry and blackberry diseases and insects (pp. 34–36). St. Paul: The American Phytopathological Society Press.Google Scholar
- Zhang, X., Zhang, G. M., Zhou, D. Q., Cheng, Y. H., Wang, Y., & Chen, M. A. (2012). A multiplex PCR and denaturing high-performance liquid chromatography method for detecting three quarantine Phytophthora species. In J. Z. Guo & B. D. Li (Eds.), Proceeding of the annual meeting of Chinese Society of Plant Pathology (pp. 50–51). Beijing: China Agricultural Science and Technology Press.Google Scholar
- Zhu, L. H., Guo, J. Z., Liao, F., Luo, J. F., Huang, G. M., Ren, X. Y., & Li, G. R. (2015). Simultaneous triplex-PCR detection of two quarantine fungal pathogens of citrus, Phytophthora hibernalis and Phytophthora syringae. Journal of Southwest University, 37(5), 1–8.Google Scholar
- Zhu, L. H., Liao, F., Cao, B. H., Li, B. J., Zhang, D. D., Luo, J. F., & Li, G. R. (2017). Quadruple-PCR molecular detection of three quarantine fungal diseases of malus. Journal of Southwest University, 39(5), 7–14.Google Scholar