Among the numerous protocols that describe the extraction of DNA, those relating to the isolation of DNA from infected plants, are rare. This study describes a rapid and reliable method of extracting a high quality and quantity of DNA from rhododendron leaves artificially infected with Phytophthora cactorum, P. cambivora, P. cinnamomi, P. citrophthora, and P. plurivora. The use of the modified Doyle and Doyle protocol (1987) allowed us to obtain high quantity and quality DNA (18.26 μg from 100 mg of the fresh weight of infected leaves at the ratios of A 260/280 and A 260/230 – 1.83 and 1.72, respectively), suitable for conventional polymerase chain reaction (PCR) and real-time PCR amplifications.
CONFLICT OF INTEREST
The authors have declared that no conflict of interests exist.
REFERENCES(26)
1.
Aljanabi S.M., Martinez I. 1997. Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. Nucleic Acids Research 25 (22): 4692–4693.
Böhm J., Hahn A., Schubert R., Bahnweg G., Adler N., Nechwatal J., Oehlmann R., Osswald 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. Journal of Phytopathology 147 (7–8): 409–416.
Caprar M., Cantor M., Sicora O., Copaci C., Sicora C. 2014. Optimization of DNA isolation from four species of Rhododendron from Europe. Journal of Horticulture, Forestry and Biotechnology 18 (1): 117–122.
Csaikl U.M., Bastian H., Brettschneider R., Gauch S., Meir A., Schauerte M., Scholz F., Sperisen C., Vornam B., Ziegenhager B. 1998. Comparative analysis of different DNA extraction protocols: A fast, universal maxi-preparation of high quality plant DNA for genetic evaluation and phylogenetic studies. Plant Molecular Biology Reporter 16 (1): 69–86.
Demeke T., Jenkins G.R. 2010. Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits. Analytical and Bioanalytical Chemistry 396 (6): 1977–1990.
Drábková L., Kirschner J., Vlček Č. 2002. Comparison of seven DNA extraction and amplification protocols in historical herbarium specimens of Juncaceae. Plant Molecular Biology Reporter 20 (2): 161–175.
Ippolito A., Schena L., Nigro F., Ligorio V.S., Yassen T. 2004. Real-time detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soil. European Journal of Plant Pathology 110 (8): 833–843.
Khanuja S.P.S., Shasany A.K., Darokar M.P., Kumar S. 1999. Rapid isolation of DNA from dry and fresh samples of plants producing large amounts of secondary metabolites and essential oils. Plant Molecular Biology Reporter 17 (1): 74.
Martin F.N., Tooley P.W., Blomquist C. 2004. Molecular detection of Phytophthora ramorum, the causal agent of sudden oak death in California, and two additional species commonly recovered from diseased plant material. Phytopathology 94 (6): 621–631.
Orlikowski L.B., Szkuta G. 2002. Occurrence of Phytophthora cinnamomi on ericaceous plants in container-grown ornamental nurseries in Poland. Journal Plant Protection Research 42 (2): 157–163.
Michiels A., Van den Ende W., Tucker M., Van Riet L., Van Laere A. 2003. Extraction of high-quality genomic DNA from latex-containing plants. Analytical Biochemistry 315 (1): 85–89.
Orlikowski L.B., Oszako T. 2005. Phytophthora cambivora on Alnus glutinosa: isolation and colonisation of plants. Journal Plant Protection Research 45 (4): 267–272.
Orlikowski L.B., Ptaszek M., Snopczyńska K. 2010. Różanecznik – nowa roślina żywicielska dla Phytophthora citrophthora w polskich szkółkach. [Rhododendron – new host plant for Phytophthora citrophthora in Polish nurseries]. Zeszyty Problemowe Postępów Nauk Rolniczych 554: 165–170. (in Polish).
Porebski S., Bailey L.G., Baum B.R. 1997. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Molecular Biology Reporter 15 (1): 8–15.
Sahu S.K., Thangaraj M., Kathiresan K. 2012. DNA extraction protocol for plants with high levels of secondary metabolites and polysaccharides without using liquid nitrogen and phenol. International Scholarly Research Network, International Scholarly Research Notices (ISRN) Molecular Biology. DOI: 10.5402/2012/205049.
Terry C.F., Harris N., Parkes H.C. 2002. Detection of genetically modified crops and their derivatives: critical steps in sample preparation and extraction. Journal of AOAC International 85 (3): 768–774.
Themann K., Werres S., Luttmann R., Dienner H.-A. 2002. Observation of Phytophthora in water recirculation systems in commercial hardy ornamental nursery stock. European Journal of Plant Pathology 108 (4): 337–343.
Weishing K., Nybom H., Wolff K., Meyer W. 1994. DNA isolation and purification. p. 44–59. In: “DNA Fingerprinting in Plants and Fungi” (K. Weising, H. Nybom, M. Pfenninger, K. Wolff, W. Meyer, eds.). CRC Press, Florida, USA, 336 pp.
Wiejacha K., Szkuta G., Orlikowska T. 2002. Optimization of DNA isolation procedure as the first step in identification of Phytophthora spp. Bulletin of the Polish Academy of Sciences, Biological Sciences 50 (3): 165–171. www.qiagen.com [Accessed: May 7, 2012].
We process personal data collected when visiting the website. The function of obtaining information about users and their behavior is carried out by voluntarily entered information in forms and saving cookies in end devices. Data, including cookies, are used to provide services, improve the user experience and to analyze the traffic in accordance with the Privacy policy. Data are also collected and processed by Google Analytics tool (more).
You can change cookies settings in your browser. Restricted use of cookies in the browser configuration may affect some functionalities of the website.