Development of seven novel specific SCAR markers for rapid identification of Phytophthora sojae: the cause of root- and stem-rot disease of soybean
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Phytophthora sojae is a devastating pathogen that causes soybean Phytophthora root and stem rot. In this study, we developed seven pairs of polymerase chain reaction primers derived from sequence-characterized amplified regions (SCAR). These seven SCAR markers allowed discrimination of P. sojae from 17 different Phytophthora species and three other soilborne pathogens (Pythium ultimum, Fusarium solani and Rhizoctonia sp.) which also induce root rot in soybean. Among those 17 Phytophthora species, P. melonis has approximately 98% similarity in ITS sequences; P. drechsleri requires an annealing temperature up to 66 °C with an ITS-targeting diagnostic marker (PS primers) developed by Wang et al. (2006) for P. sojae; and P. sansomeana is a newly described soybean-infecting Phytophthora species. These three Phytophthora species could be specifically distinguished against P. sojae by these seven SCAR markers. After screening 100 random amplified polymorphic DNA (RAPD) primers, eight primers clearly produced specific bands only for P. sojae rather than other Phytophthora species tested. Subsequently, seven of eight P. sojae-specific RAPD markers were successfully converted into SCAR markers, namely, Scar276, Scar304, Scar333, Scar37, Scar519, Scar57 and Scar78. These SCAR markers were used to detect 75 isolates of P. sojae specifically, while no products were obtained for 29 additional isolates representing 17 other Phytophthora species and three other soilborne pathogens. Furthermore, Scar333 successfully allowed the detection with a sensitivity of 100 pg from genomic DNA of P. sojae, Scar276 had a higher sensitivity of 10 pg, and four specific SCAR primers (Scar304, Scar37, Scar519 and Scar78) had a sensitivity of 100 fg, which is the highest for detecting P. sojae until now. Six of the seven SCAR markers, with the exception of Scar57, were also used to detected P. sojae in artificial or naturally infected soybean seedlings and infested soil. Our findings demonstrate that SCAR markers provide a rapid and sensitive molecular diagnostic tool for the detection of P. sojae in plants, and will play a key role in effective management of the disease.
KeywordsPhytophthora sojae Molecular detection RAPD Root rot SCAR marker
This research was partly supported by grants provided to Qin Xiong by the National Natural Science Foundation of China (No. 31600512) and the Natural Science Foundation of Jiangsu Province (BK20160923), to Yuanchao Wang by Special Fund for Agro-scientific Research in the Public Interest (201303018), to Chen Zhang, Yu Zhu and Xinyue Zheng by Students Practice Innovation and Training Program of Nanjing Forestry University (201710298045Z, 201710298064Z, 2016NFUSPITP206) respectively, and by the Priority Academic Program Development of Jiangsu Higher Education Institutions. The authors declare that they have no conflict of interest. We highly appreciate W. H. Ko (Hawaii University), Brett Tyler (Oregon State University), J. H. Peng (Dalian Quarantine Bureau) and Q. H. Tang (Nanjing Agricultural University) for providing us with the isolates of Phytophthora spp. and other pathogens. We also thank James T. Wong (University of California Riverside), Peng Wang (University of California Riverside), Justin Waletich (Oregon State University) and Kai Tao (Oregon State University) for their editorial assistance.
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Conflict of interest
The authors declare that they have no conflict of interest in the submission of this manuscript;
This article does not contain any studies with human participants or animals performed by any of the authors.
Informed consent was obtained from all individual participants included in the study. All authors in this manuscript have read and approved current version of this article;
The manuscript has not been submitted to more than one journal for simultaneous consideration.
- Bulat, S. A., Lübeck, M., Alekhina, I. A., Jensen, D. F., Knudsen, I. M., & Lübeck, P. S. (2000). Identification of a universally primed-PCR-derived sequence-characterized amplified region marker for an antagonistic strain of Clonostachys rosea and development of a strain-specific PCR detection assay. Applied and Environmental Microbiology, 66, 4758–4763.CrossRefGoogle Scholar
- Chen, X., & Wang, Y. (2017). Phytophthora sojae. In Biological invasions and its Management in China (pp.199–223): Springer.Google Scholar
- Erwin, D. C., & Ribeiro, O. K. (1996). Phytophthora diseases worldwide. American Phytopathological Society. APS Press.Google Scholar
- Gotor-Vila, A., Teixidó, N., Usall, J., Dashevskaya, S., & Torres, R. (2016). Development of a SCAR marker and a strain-specific genomic marker for the detection of the biocontrol agent strain CPA-8 Bacillus amyloliquefaciens (formerly B. subtilis). Annals of Applied Biology, 169, 248–256.CrossRefGoogle Scholar
- Hansen, Z. R., Knaus, B. J., Tabima, J. F., Press, C. M., Judelson, H. S., Grünwald, N. J., & Smart, C. D. (2016). Loop-mediated isothermal amplification (LAMP) for detection of the tomato and potato late blight pathogen, Phytophthora infestans. Journal of Applied Microbiology, 120, 1010–1020.CrossRefGoogle Scholar
- Kaluzna, M., Albuquerque, P., Tavares, F., Sobiczewski, P., & Pulawska, J. (2017). Development of SCAR markers for rapid and specific detection of Pseudomonas syringae pv. morsprunorum races 1 and 2, using conventional and real-time PCR. Applied Microbiology and Biotechnology, 101, 903–903.CrossRefGoogle Scholar
- Liu, Y., Chen, X., Jiang, J., Hamada, M. S., Yin, Y., & Ma, Z. (2014). Detection and dynamics of different carbendazim-resistance conferring β-tubulin variants of Gibberella zeae collected from infected wheat heads and rice stubble in China. Pest Management Science, 70, 1228–1236.CrossRefGoogle Scholar
- Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual (Vol. 2): Cold Spring Harbor Laboratory Press New York.Google Scholar
- Scauflaire, J., Godet, M., Gourgue, M., Liénard, C., & Munaut, F. (2012). A multiplex real-time PCR method using hybridization probes for the detection and the quantification of Fusarium proliferatum, F. subglutinans, F. temperatum, and F. verticillioides. Fungal Biology, 116, 1073–1080.CrossRefGoogle Scholar
- Schena, L., Li Destri Nicosia, M., Sanzani, S., Faedda, R., Ippolito, A., & Cacciola, S. (2013). Development of quantitative PCR detection methods for phytopathogenic fungi and oomycetes. Journal of Plant Pathology, 95, 7–24.Google Scholar
- Schmitthenner, A. (1999). Phytophthora rot of soybean. In Compendium of Soybean Diseases. 4th edn.(pp.39–42). St. Paul, Minnesota: The American Phytopathological Society Press.Google Scholar
- Su, Y., & Shen, C. (1993). The discovery and biological characteristic studies of Phytophthora megasperma f. Sp. glycinea on soyabean in China. Acta Phytopathologica Sinica, 23, 341–347.Google Scholar
- Sugimoto, T., Kato, M., Yoshida, S., Matsumoto, I., Kobayashi, T., Kaga, A., Hajika, M., Yamamoto, R., Watanabe, K., Aino, M., Matoh, T., Walker, D. R., Biggs, A. R., & Ishimoto, M. (2012). Pathogenic diversity of Phytophthora sojae and breeding strategies to develop Phytophthora-resistant soybeans. Breeding Science, 61, 511–522.CrossRefGoogle Scholar
- Vu, N. T., Pardo, J. M., Alvarez, E., Le, H. H., Wyckhuys, K., Nguyen, K. L., et al. (2016). Establishment of a loop-mediated isothermal amplification (LAMP) assay for the detection of phytoplasma-associated cassava witches’ broom disease. Applied Biological Chemistry, 59, 151–156.CrossRefGoogle Scholar
- Yadav, M. K., & Singh, B. P. (2017). Real-time polymerase chain reaction (PCR) based identification and detection of fungi belongs to genus Fusarium. In Molecular Markers in Mycology (pp.65–85): Springer.Google Scholar
- Zheng, X. (1995). Methods in Phytophthora. Beijing: Chinese Agriculture Press.Google Scholar