Quadruplex PCR detection of three quarantine Phytophthora pathogens of berries

  • Fang Liao
  • Guo-Ming Huang
  • Lin-Hui Zhu
  • Dajin Lv
  • Doudou Zhang
  • Jia-Feng Luo
  • Guan-Rong LiEmail author


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.


Phytophthora 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

Ethical statement

This research did not involve any animal and/or human participants. The authors declare that they have no conflict of interests.


  1. Amouzou-Alladaye, E., Dunez, J., & Clerjeau, M. (1988). Immunoenzymatic detection of Phytophthora fragariae in infected strawberry plants. Phytopathology, 78, 1022–1026.CrossRefGoogle Scholar
  2. Anderson, H. W. (1935). Black stele root rot of strawberry. Phytopathology, 25, 5.Google Scholar
  3. Bonants, P., de Weerdt, M. H., van Gent-Pelzer, M., Lacourt, I., Cooke, D., & Duncan, J. (1997). Detection and identification of Phytophthora fragariae Hickman by the polymerase chain reaction. European Journal of Plant Pathology, 103(4), 345–355.CrossRefGoogle Scholar
  4. Burns, R., & George, E. (1995). The use of monoclonal antibodies for the detection of fungi. Bulletin OEPP/EPPO Bulletin, 25, 31–38.CrossRefGoogle Scholar
  5. Chen, Y., & Roxby, R. (1996). Characterization of a Phytophthora infestans gene involved in the vesicle transport. Gene, 181(1/2), 89–94.CrossRefGoogle Scholar
  6. Cooke, D. E. L., Duncan, J. M., & Unkles, S. (1995). Diagnosis and detection of Phytophthora fragariae in raspberry and strawberry. Bulletin OEPP/EPPO Bulletin, 25, 95–98.CrossRefGoogle Scholar
  7. Dominik, S., Michał, O., Magdalena, S., & Grzegorz, B. (2015). Genetic diversity of Trichoderma atroviride strains collected in Poland and identification of loci useful in detection of within-species diversity. Folia Microbiologica, 60(4), 297–307.CrossRefGoogle Scholar
  8. 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
  9. EPPO (2018) EPPO Global Database (available online).
  10. 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
  11. Gao, R., & Zhang, G. (2013). Potential of DNA barcoding for detecting quarantine fungi. Phytopathology, 103, 1103–1107.CrossRefGoogle Scholar
  12. Gao, R., Cheng, Y., Wang, Y., Wang, Y., Guo, L., & Zhang, G. (2015). Genome sequence of Phytophthora fragariae var. fragariae, a quarantine plant-pathogenic fungus. Genome Announcements, 3(2), e00034–e00015. Scholar
  13. Henegariu, O., Heerema, N. A., Dlouhy, S. R., Vance, G. H., & Vogt, P. H. (1997). Multiplex PCR: Critical parameters and step-by- step protocol. BioTechniques, 23(3), 504–511.CrossRefGoogle Scholar
  14. 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
  15. Ippolito, A., Schena, L., & Nigro, F. (2002). Detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soils by nested PCR. European Journal of Plant Pathology, 108(9), 855–868.CrossRefGoogle Scholar
  16. 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
  17. Kroon, L. P., Brouwer, H., de Cock, A. W., & Govers, F. (2012). The genus Phytophthora anno 2012. Phytopathology, 102, 348–364.CrossRefGoogle Scholar
  18. 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
  19. Langrell, S. R. H., Morel, O., & Robin, C. (2011). Touchdown nested multiplex PCR detection of Phytophthora cinnamomi and P. cambivora from French and English chestnut grove soils. Fungal Biology, 115(7), 672–682.CrossRefGoogle Scholar
  20. Li, M. Z., Asano, T., Suga, H., & Kageyama, K. (2011). A multiplex PCR for the detection of Phytophthora nicotianae and P. cactorum, and a survey of their occurrence in strawberry production areas of Japan. Plant Disease, 95(10), 1270–1278.CrossRefGoogle Scholar
  21. Liu, Z. Y., Kang, Y. P., & Yuan, B. (2008). Identification of Phytophthora fragariae. Plant Protection, 34(5), 163–165.Google Scholar
  22. 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
  23. Man in’t Veld, W. A. (2007). Gene flow analysis demonstrates that Phytophthora fragariae var. rubi constitutes a distinct species, Phytophthora rubi comb. nov. Mycologia, 99(2), 222–236.CrossRefGoogle Scholar
  24. McKeen, W. E. (1958). Red stele root disease of the loganberry and strawberry caused by Phytophthora fragariae. Phytopathology, 48, 129–132.Google Scholar
  25. Mohan, S. B. (1988). Evaluation of antisera raised against Phytophthora fragariae for detecting the red core disease of strawberries by enzyme-linked immunosorbent assay (ELISA). Plant Pathology, 37, 206–216.CrossRefGoogle Scholar
  26. Pepin, H. S. (1967). Susceptibility of members of the Rosaceae to races of Phytophthora fragariae. Phytopathology, 57, 782–784.Google Scholar
  27. Pscheidt, J. W., Burket, J. Z., Fischer, S. L., & Hamm, P. B. (1992). Sensitivity and clinical use of Phytophthora-specific immunoassay kits. Plant Disease, 76, 928–932.CrossRefGoogle Scholar
  28. Stammler, G., Seemüller, E., & Duncan, J. M. (1993). Analysis of RFLPs in nuclear and mitochondrial DNA and the taxonomy of Phytophthora fragariae. Mycological Research, 97, 150–156.CrossRefGoogle Scholar
  29. 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
  30. Wardlaw, C. W. (1927). The strawberry disease in Lanarkshire. Annals of Applied Biology, 14(2), 197–201.CrossRefGoogle Scholar
  31. 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
  32. 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
  33. Wilcox, W. F., & Duncan, J. M. (1993). Phytophthora fragariae Hickman var. rubi var. nova. Mycological Research, 97, 830.CrossRefGoogle Scholar
  34. Wilcox, W. F., Scott, P. H., Hamm, P. B., Kennedy, D. M., Duncan, J. M., Brasier, C. M., & Hansen, E. M. (1993). Identity of a Phytophthora species attacking raspberry in Europe and North America. Mycological Research, 97, 817–831.CrossRefGoogle Scholar
  35. 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
  36. 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
  37. 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

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2019

Authors and Affiliations

  • Fang Liao
    • 1
    • 2
  • Guo-Ming Huang
    • 2
  • Lin-Hui Zhu
    • 1
  • Dajin Lv
    • 1
  • Doudou Zhang
    • 1
  • Jia-Feng Luo
    • 2
  • Guan-Rong Li
    • 1
    Email author
  1. 1.College of Agronomy and BiotechnologySouthwest UniversityChongqingChina
  2. 2.Tianjin Entry-Exit Inspection and Quarantine BureauTianjinChina

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