Problems Encountered with Nested PCR to Diagnosis of Phytoplasmas

Document Type : Research Article


1 Institute Tarbiat modarres/ Seed and Plant Certification and Registration Istitute

2 Ferdowsi University of Mashhad


Introduction: Phytoplasmas are phytopathogenic bacteria without cell walls that can be found in plant phloem, have been found associated with numerous diseases of plants worldwide. A sensitive and precise diagnostic test for detection of phytoplasma-infected plant is critical to avoid using infected planting material and dispersal of these agents. The detection of phytoplasma from plant tissues by PCR, requires using DNA extraction methods that extract high level phytoplasmas DNA with less plant inhibitors. Usually Phytoplasma diagnostics have been based on the 16S rRNA gene and the 16S–23S rRNA spacer region because universal primers that design for replication these regions could detect different groups of phytoplasmas but diagnostics based on these primers can be problematic, with occasional false positives, through amplification of some bacterial genomes that might be present in a plant sample. These primers also have sequence homology to chloroplasts and plastids and increase the risk of false positives so it is important to guard against false negatives and positive during such detection techniques.
Materials and Methods: Health and infected Lime samples with Candidatus phytoplasma aurantifolia were used respectively as negative and positive samples. DNA was extracted by the CTAB methods, SDS method, Fermentas DNA extraction kit, column based Method (Genet Bio genomic DNA isolation kit) and compared to remove inhibitors and reduced false negative reactions in nested PCR detection method. DNA samples were tested for phytoplasma infection by direct PCR using the universal phytoplasma primer pair P1/P7 and nested PCR using primer pairs P1/P7-R16F2n/R16R2 and P1/P7-fU5/rU3. The PCR products were sequenced and subsequent analysis using GenBank database information at the national center for biotechnology was employed.
Results and Discussion: Comparison of different DNA extraction methods indicated using suitable method can significantly reduce false negative reaction, but even in successful column-based DNA extraction method, false negative reactions were reported that were due to low phytoplasma concentration and irregular distribution within host tissues or could be caused by inhibitor presence in DNA samples. Based on results of sequencing, false positives were obtained sporadically, using primer pairs combination P1/P7- R16F2n/R16R2 that may be arising from cross over contamination or sequence homology with plant genome, so some primers can react probably with sequences of plant genome and false positives could be observed. Since false positives are also a major problem in PCR protocols, especially in nested PCR so single tube nested PCR (STNP) was optimized to avoid false positive reaction but regardless advantages of this method such as facility, cost and time effective and ability to detect low concentration of pathogen, false positive reactions were observed in a few samples. The advantage of STNP is that tubes do not have to be opened, so the risk of contamination minimized.
Conclusion: PCR-based techniques for phytoplasma detection, appears to be the method of choice because of their high sensitivity and specificity. Using suitable method for extraction of DNA from infected plant tissues are getting more critical for precise detection through increasing DNA quality and quantity. In the other hand confirmation of phytoplasma presence must be accomplished at least by RFLP analyses or different primer pair combination to avoid false positive detection.


1- Ahrens U. and Seemuller E. 1992. Detection of DNA of plant pathogenic mycoplasma like organisms by a polymerase chain reaction that amplifies a sequence of the 16S rRNA gene. Phytopathology, 82: 828-832.
2- Berges R., Rott M. and Seemüller E. 2000. Range of phytoplasma concentrations in various plant hosts as determined by competitive polymerase chain reaction. Phytopathology, 90:1145–1152
3- Borah B.K., Johnson A., Sai Gopal D.V.R. and Dasgupta I. 2008. A comparison of four DNA extraction methods for the detection of Citrus yellow mosaic badna virus from two species of citrus using PCR and dot-blot hybridization. Journal of Virological Methods, 151: 321-324.
4- Cankar K., Stebih D., Dreo T., Ţel J. and Gruden K. 2006. Critical points of DNA quantification by real-time PCR – Effects of DNA extraction method and sample matrix on quantification of genetically modified organisms. BMC Biotechnology, 6: 37-51.
5- Christensen N.M., Nyskjold H. and Nicolaisen, M. 2013. Real-time PCR for universal phytoplasma detection and quantification. Methods in Molecular Biology, 938:245-52.
6- Davis R.E. and Lee M. 1993. Cluster-specific polymerase chain reaction amplification of 16S rDNA sequences for detection and identification of mycoplasma like organisms. Phytopathology, 83: 1008-1001.
7- Davis T.M., Yu H. Haigis K.M. and McGowan P.J. 1995. Template mixing: A method of enhancing detection and interpretation of codominant RAPD markers. Theoretical and Applied Genetics, 91:582-588
8- Deng S.J. and Hiruki C. 1991. Amplification of 16S ribosomal-RNA genes from culturable and nonculturable mollicutes. Journal of Microbiological Methods, 14:53–61
9- Doyle J.J. and Doyle J.L. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical bulletin, 19:11-15.
10- Franova J. 2011. Difficulties with conventional phytoplasma diagnostic using PCR/RFLP analyses. Bulletin of Insectology, 64: 287-288
11- Gundersen D.E. and Lee I.M. 1996. Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer sets. Phytopathologia Mediterranea, 35: 144-151.
12- Harrison N.A., Griffiths H.M., Carpio M.L. and Richardson P.A. 2001. Detection and characterization of an elm yellows (16SrV) group phytoplasma infecting Virginia creeper plants in southern Florida. Plant Disease, 85: 1055–1062
13- Heinrich M., Botti SCaprara L., Arthofer W. and Strommer S. 2001. Improved detection methods for fruit tree phytoplasmas. Plant Molecular Biology Reporter, 19: 169-179.
14- Javaheri M., Ghareyazie B., Delkhosh H.R. and Rahimian H. 2000. Detection of witches’ broom infected citrus trees using polymerase chain reaction. P.147. Proceeding of 14th Iranian Plant Protection Congress. 5-8 Sept. 2000, Esfehan. Iran. (in Persian with English abstract).
15- Jiang J., Aldeisio A.K., Singh A. and Xiao L. 2005. Development of Procedures for Direct Extraction of Cryptosporidium DNA from Water Concentrates and for Relief of PCR Inhibitors. Applied and Environmental Microbiology, 71: 1135–1141.
16- Lee I.M. and Davis R.E. 1986. Prospects for in vitro culture of plant-pathogenic mycoplasma like organisms. Annual Review of Phytopathology. 24:339-354.
17- Lee I-M., Davis R.E. and Gundersen-Rindal D.E. 2000. Phytoplasma: phytopathogenic mollicutes. Annual Review of Microbiology, 54:221–255
18- Lee I-M, Hammond R.W., Davis R.E. and Gundersen D.E. 1993. Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasmalike organisms. Phytopathology, 83: 834-842.
19- Lepka P., Stitt M., Moll E. and Seemuller E. 1999. Effect of phytoplasmal infection on concentration and traslocation of carbohydrates and amino acids in periwinkle and tobacco. Physiological and Molecular Plant Pathology, 55: 59–68.
20- Leyva-Lopez NE, Ochoa-Sanchez JC, Leal-Klevezas DS, Martinez-Soriano JP. (2002). Multiple phytoplasmas associated with potato diseases in Mexico. Canadian Journal of Microbiology, 48: 1062-1068.
21- Liop P., Bonaterra A., Peñalver J. and Lopez M.M. 2000. Development of a highly sensitive nested-PCR procedure using a single closed tube for detection of Erwinia amylovora in asymptomatic plant material. Applied and Environmental Microbiology, 66: 2071- 2078.
22- Lorenz K.H., Schneider B., Ahrens U. and Seemuller E. 1995. Detection of the apple proliferation and pear decline phytoplasmas by PCR amplification of Ribosomal and non-ribosomal DNA .Phytopathology, 85:771-776
23- McGarvey P. and Kaper J.M. 1991. A simple and rapid method for screening transgenic plants using the PCR. BioTechniques, 11:428–432.
24- Marzachi C. 2004. Molecular diagnosis of phytoplasmas. Phytopathologia Mediterranea, 43: 228-231.
25- Mayr W., Winidhorst S. and Hillemeier K. 2005. Antiproliferative plant and synthetic polyphenolics are specific inhibitors of vertebrate inositol-1,4,5-trisphosphate 3-kinases and inositol polyphosphate multikinase. Journal of Biological Chemistry, 280: 13229–13240.
26- Mirzaee M., Heydarnejad J., Saleh M. Hosseini Pour A., Massumi H. and Shabanian M. 2010. production of polyclonal antiserum against the causal agent of lime witches'-broom. Iranian Journal of Plant Pathology, 45:155- 159. (in Persian with English abstract).
27- Moslemkhani C., Razavi V., Sadeghi L, Mobasser S, Khelgati bana F. and Shahbazi R. 2014. Characterization of phytoplasmas associated with tomato big bud disease, using single-tube nested PCR. Journal Crop Protection, 3:573-580
28- Musetti R., Favali A. and Pressacco L. 2000. Histopathology and polyphenol content in plants infected by phytoplasmas. Cytobios, 102: 133–147.
29- Nolan T., Hands R.E., Ogunkolade W. and Bustin S. 2006. SPUD: A quantitative PCR assay for the detection of inhibitors in nucleic acid preparation. Analytical Biochemistry, 351: 308-310.
30- Pastrik K.H. and Maiss E. 2000. Detection of Ralstonia solanacearum in potato tubers by polymerase chain reaction. Journal of Phytopathology, 148: 619-626.
31- Sadeghi A., Salehi M., Faghihi M.M., Bagheri A.N. and Samavi S. 2010. Detection of witches'broom disease of lime phytoplasma in crown region by PCR during latentperiod of the disease. p. 517. Proceeding of 19th Iranian Plant Protection Congress, 31 Jul -3 Aug. 2010, Tehran, Iran. (in Persian with English abstract)
32- Shahryari F., Shams-Bakhsh M., Safarnejad M.R., Safaie N. and Ataei Kachoiee S. 2013. Preparation of Antibody against Immunodominant Membrane Protein (IMP) of Candidatus Phytoplasma aurantifolia. Iranian Journal of Biotechnology 11: 14-21.
33- Siampour M., Izadpanah K., Afsharifar A.R. Salehi M. and Taghizadeh M. 2006. Detection of phytoplasma in insects collected in witches′ broom affected lime groves. Iranian Journal of Plant Pathology, 42: 139-158. (in Persian with English abstract)
34- Wally V., Klausegger A., Koller U., Lochmuller H., Krause S., Wiche G., Mitchell L.G., Hintner H. and Bauer J.W. 2008. 5' trans-splicing repair of the PLEC1 gene. Journal of Investigative Dermatology. 128 (3): pp. 568-574.
35- Wei T., Lu G. and Clover G. 2008. Novel approaches to mitigate primer interaction and eliminate inhibitors in multiplex PCR, demonstrated using an assay for detection of three strawberry viruses. Journal of Virological Methods, 151: 132-139.
36- Zhang Y P, Uymoto J K, and Kirkpatrick B C. (1998) A small-scale procedure for extracting nucleic acids from woody plants infected with various phytoplasmas for PCR assay. Journal of Virological Methods, 71: 45-50.