Triplex real-time PCR detection of three quarantine Phytophthora pathogens infecting Malus Miller
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In order to develop a simultaneous and specific molecular detection of the three quarantine Phytophthora pathogens, Phytophthora hibernalis, Phytophthora cambivora and Phytophthora syringae that infect Malus Miller, three pairs of real-time PCR primers (PH-F/PH-R, PC-F/PC-R and PS-F/PS-R) and three probes (PH-Pr, PC-Pr and PS-Pr) labeled with HEX, FAM and ROX, respectively, were designed for P. hibernalis, P. cambivora and P. syringae by alignment analyses of enolase (Enol), ras-like protein Ypt1 and HSP90 gene sequences with other Phytophthora spp. Black Hole Quencher 1 (BHQ1) was used for P. hibernalis and P. cambivora and BHQ2 for P. syringae. Through the optimization of the reaction conditions, a triplex real-time PCR simultaneous detection for the three Phytophthora species infecting Malus Miller was developed. It could achieve simultaneous and specific detection with a sensitivity of 2 × 10−4, 2 × 10−4 and 2 × 10−2 ng/μL genomic DNA, respectively, for P. hibernalis, P. cambivora and P. syringae.
KeywordsMalus Miller Quarantine Phytophthora pathogens P. hibernalis P. cambivora P. syringae Triplex real-time PCR detection
This work was funded by Rapid Identification of Quarantine Fungal Phytophthora Pathogens of Imported Fruits and Seedlings, a Program from The General Administration of Quality Supervision, Inspection and Quarantine of PRC (No: 2012IK286) and Plant Quarantine Pathogen Detection and Application by DNA Barcoding, National “12th Five-Year” Science and Technology Support Program (No: 2012BAK11B02).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interests.
This research did not involve any animal and/or human participants.
- Böhm J, Hahn A, Schubert R, Bahnweg G, Adler N, Nechwatal J, Oehlmann R, Obszwald W (1999) Real-time quantitative PCR: DNA determination in isolated spores of the Mycorrhizal Fungus Glomus mosseae and monitoring of Phytophthora infestans and Phytophthora citricola in their respective host plants. J Phytopathol 147(7/8):409–416CrossRefGoogle Scholar
- Cubero J, Ayllon MA, Gell I, Melgarejo P, De Cal A, Martín-Sánchez PM, Pérez-Jiménez RM, Soria C, Segundo E, and Larena I. (2009) Detection of Strawberry pathogens by real-time PCR. In: Proceedings of VIth Internat. Strawberry SymposiumGoogle Scholar
- Du HZ, Wu PS, Yan J, Zhang QW (2013) Detection of Phytophthora hibernalis by real-time fluorescent PCR. Plant Quar 27(1):36–39Google Scholar
- Feng LP, Liu HM, Wang YC, Li Y, Wu XH, Ji Y, Wu PS (2011) SN/T 2759–2011. The Entry-exit Inspection and Quarantine Industry Standards of the People’s Republic of China–Quarantine and Identification of Phytophthora cambivora (Petri) Buisman. China Standards Press, BeijingGoogle Scholar
- Fujita K, Nakazawa N, Fukushima C, Harada Y (1994) Phytophthora fruit rot of apple, Japanese pear and European pear caused by Phytophthora syringae (Kleb). Ann Phytopathol Soc Jpn 6(6):717–724Google Scholar
- Gao QF, Qin XY, Fang DH, Mo XH, Yang GH, Chen HR, Cai H (2012) A rapid method to detect Phytophthora nicotianae by PCR. J Yunnan Agric Univ 27(2):156–159Google Scholar
- Liu Y, Zhu L, Li P, Liao F, Ren X, Li G (2015) Triplex-PCR molecular detection of two quarantine fungal diseases of Prunus-Phytophthora syringae and P. cambivora. J Plant Prot 42(4):571–577Google Scholar
- Luo JF, Liu YT, Liao F, Hu XH, Liu P, Huang MG (2012) Interception of Phytophthora syringae on citrus fruits imported from California, USA. Mycosystema 31(1):24–30Google Scholar
- Qian GZ, Tang GG (2005) A review on the plant taxonomic study on the genus Malus Miller. J Nanjing For Univ 29(3):94–98Google Scholar
- Schena L, Nigro F, Ippolito A (2004) Real-time PCR detection and quantification of Soilborne Fungal pathogens: the case of Rosellinia necatrix, Phytophthora nicotianae, P. citrophthora, and Verticillium dahliae. Phytopathol Mediterr 43:273–280Google Scholar
- Wang H, Zhu RL, Tan YL, Wei K, Wang XJ, Sun ZH, Sheng PC (2011a) Establishment and application of multiple PCR for diagnosing Proteus mirabilis, Salmonella and Listeria monocytogenes. Sci Agric Sin 44(11):2334–2340Google Scholar
- Wang YC, Li Y, Wu PS, Shao XL, Zhang JX, Yu DD, Gan QH, Song T (2011b) SN/T 2756–2011. The Entry-exit Inspection and Quarantine Industry Standards of the People’s Republic of China-Inspection and Identification of Phytophthora syringe (Berk) Kleb. China Standards Press, BeijingGoogle Scholar
- Wilcox WF (1993) Incidence and severity of crown and root rots on four apple rootstocks following exposure to Phytophthora species and waterlogging. J Am Soc Hortic Sci (JASHS) 118(1):63–67Google Scholar
- Wu PS, Du HZ, Yan J (2010) SN/T 2617–2010. The Entry-exit Inspection and Quarantine Industry Standards of the People’s Republic of China–Detection and Identification of Phytophthroa hibernalis Carne. China Standards Press, BeijingGoogle Scholar
- Zhang HF, Ren Z, Liu X, Zhang ZG, Wang YC, Wu XH, Zheng XB (2008) Rapid molecular detection of Phytophthora hilbernalis by PCR. Acta Phytopathol Sin 38(3):231–237Google Scholar
- Zhuc Linhui, Guo Jingze, Liao Fang, Luo Jiafeng, Huang Guoming, Ren Xueyi, Li Guanrong (2015) Simutaneous triplex PCR detection of two quarantine fungal pathogens of Citrus, Phytoghthora hiberalis and Phytoghthora syringae. J Southwest Univ 37(5):1–8Google Scholar