Detection of sandal spike phytoplasma by polymerase chain reaction

Sunil Thomas and M. Balasundaran*

Division of Pathology, Kerala Forest Research Institute, Peechi, Thrissur 680 653, India

Spike disease affected sandal (Santalum album L.) tissues were screened for the presence of the pathogen, a non-culturable phytoplasma using polymerase chain reaction (PCR) technique. Oligonucleotide primers specific to the conserved region of 16S rRNA gene were used to amplify a 558 bp sequence of the phytoplasma. Four DNA fragments were obtained when the PCR products after 20 cycles of amplification were subjected to restriction fragment length polymorphism analysis (RFLP) with AluI restriction endonuclease. The technique confirms that sandal spike phytoplasma belongs to group I of the eleven major phytoplasma groups.

SPIKE disease, the major disease in sandal (Santalum album L.), is caused by a non-culturable phytoplasma seen exclusively in the phloem tissues1. Detection of the pathogen was mainly through electron microscopy2–4 or indirect methods using different stains like aniline blue5 and Giemsa6 stain by light microscopy. Recently, the DNA binding fluorochrome, 4,6 diamidino-2-phenylindole (DAPI) was employed for the detection of the pathogen7. Since phytoplasmas are associated with diseases of many plant species8, neither of these methods allows the differentiation of the organism and its classification9.

Studies of DNA homology in the highly conserved genes encoding ribosomal RNA and ribosomal protein have shown that phytoplasmas comprise a coherent set distinct from other prokaryotes10,11. A system for classification of phytoplasma based on amplification of the 16S rDNA by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) was introduced by Ahrens and Seemuller9. Since then, several groups of phytoplasmas have been differentiated on the basis of nucleotide sequence variation in 16S rRNA genes11–13. Sinclair et al.14 have classified phytoplasmas into eleven major taxonomic groups based on the 16S RFLP fragments. In this paper we report the detection of phytoplasma from spike disease affected sandal by PCR using group specific primer followed by RFLP analysis.

Spike disease affected sandal was collected from Marayoor, Munnar Forest Division, Kerala and
Chamundi Hills, Mysore, Karnataka. Total DNA samples from healthy and diseased plants were extracted


*For correspondence.

following the modified protocol of Doyle and Doyle15. One gram tissue of midrib and young stem was ground into fine powder using liquid nitrogen. Two ml of hot (65oC) cetyl trimethyl ammonium bromide (CTAB) buffer (2% CTAB, 100 mM Tris, pH 8.0; 20 mM EDTA, pH 8.0; 1.4M NaCl) was added to the powder and samples incubated at 65oC for 1 h. The samples were extracted with chloroform/isoamyl alcohol (24:1) followed by low speed centrifugation (1600 g) for 5 min and aqueous phase eluted out. To this was added 1/10 volume 3M sodium acetate (pH 5.2) followed by the addition of 2 volumes of cold (–20oC) absolute alcohol. After incubation at –20oC for 12 h, the DNA precipitate was centrifuged for 10 min at low speed. Ethanol (95%) was added to the DNA pellet and recentrifuged at the same speed. The supernatant was discarded, air dried and the pellet dissolved in 100 m l sterile distilled water. The total DNA was estimated at A260 (1 O.D = 50 m g ml–1).

The method followed by Ahrens and Seemuller9 that allowed the amplification of a 558 bp fragment of the 16S rRNA gene of phytoplasma was adopted for PCR with slight modification. The primers were designed from the conserved regions of the 16 S rRNA gene of O-MLO16 located between 759 and 1359 bp. The sequence of the forward primer is 5 -ACGAAAGCGTG-GGGAGCAAA-3 and the reverse primer is 5 -GAAGTCGAGTTGCAGACTTC-3 . A total volume mixture of 50 m l contained 1 m l of test DNA (200 ng), 1 m l of each primer (Bangalore Genei, India), 2.5 mM each of four dNTPs, 1 m l (3 units) of Taq polymerase (Bangalore Genei, India) or Dynazyme II (Finnzymes Oy, Finland) and 5 m l Taq buffer or Dynazyme buffer. The mixture was covered with two drops of mineral oil and subjected to 20 amplification cycles (PTC-150 Minicycler, MJ Research, USA) each of 30 s denaturation (95oC), 30 s annealing (55oC) and 30 s extension (72oC). The final extension step was for 5 min.

The PCR amplification products obtained from DNA of healthy and diseased sandal after 20 cycles were electrophoresed in 1.5% horizontal agarose (Sigma, USA) gel in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0). The gel was stained with ethidium bromide and bands compared with a 100 bp DNA ladder (Bangalore Genei, India). 15 m l of the reaction mixture obtained using Taq polymerase was digested with 1 m l of undiluted AluI (Bangalore Genei, India) following manufacturer’s instruction at 37oC for 12 h. 10 m l of the digest was used to resolve the restriction fragments on a 3% horizontal agarose gel in TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8.0), and the bands were visualized after staining with ethidium bromide. The gels were documented using Kodak Digital Science Electrophoresis Documentation and Analysis System 120 (Kodak, USA).

After 20 amplification cycles, the DNA fragment of 558 bp was amplified from samples which contained DNA extracted from diseased sandal but not from healthy samples or water control. Bands were visualized for both Taq polymerase and Dynazyme II polymerase (Figure 1). On comparing the spike diseased tissues from Kerala and Karnataka, the bands appeared to be identical (Figure 2). Restriction analysis of the amplified fragment with AluI revealed the presence of a pattern characterized by restriction sites at position a, b, and c with fragments of 240, 191, 71 and 56 bp (Figure 3 a, b), as described by Ahrens and Seemuller9 for the first group of phytoplasmas.

Since phytoplasma cannot be cultured under axenic conditions, their identities and taxonomic position were unclear or uncertain until recently, when methods of

1575a.jpg (39714 bytes) 

Figure 1.  Agarose gel electrophoresis of polymerase chain reaction product (16S rDNA fragment –558 bp) of sandal spike phytoplasma after 20 cycles of amplification using both Taq polymerase (lanes 2–4) and Dynazyme II (lanes 5–7). Lane 1, 100 bp ladder; lanes 2 and 5, healthy sandal; lanes 3, 4, 6 and 7, diseased sandal; lane 8, water control.

 

 

 

 

 

 

 

Figure 2.  Agarose gel electrophoresis of polymerase chain reaction product (16S rDNA fragment –558 bp) of sandal spike phytoplasma after 20 cycles of amplification using Kerala (lanes 3–5) and Karnataka (lanes 6–8) sandal spike infected tissues. Lane 1, 100 bp ladder; lane 2, healthy sandal.

 

 

1575b.jpg (25774 bytes) 

Figure 3.  a, Agarose gel electrophoresis of AluI digests of PCR products of sandal spike phytoplasma. Lane 1, 100 bp ladder; lanes 2 and 3, sandal spike phytoplasma of Kerala; lanes 4 and 5, sandal spike phytoplasma of Karnataka; b, AluI restriction maps of the 558 bp 16S rDNA fragments of group I phytoplasma (Ahrens and Seemuller9).

 

molecular genetics were introduced into plant mycoplasmology17. The 16S rRNA gene, is a universal character which provides valuable phylogenetic and taxonomic information on culturable mollicutes and other prokaryotes9,10,13. Hence, this gene has widely been used to obtain phylogenetic information on non-culturable phytoplasmas. RFLP analysis of the amplified PCR product (rDNA) could distinguish various groups and subgroups of phytoplasma17.

PCR method of detecting phytoplasmas gained popularity after the classical work of Ahrens and Seemuller9. The primers used were designed from the conserved regions of the 16S rRNA gene of the Oenothera phytoplasma (O-MLO). Phytoplasmas infecting several plants collected from different continents maintained in Catheranthus roseus for several years were used for their study. The mollicutes have a high mutation rate resulting in many totally unique oligonucleotides18. In our study, we used young stem and leaf midrib tissues from diseased and healthy sandal for isolating DNA, since the greatest yields of DNA will always be obtained using the youngest, freshest tissue. The liquid nitrogen powdered tissues were incubated at 65oC for 1 h for larger DNA yields19. 3M sodium acetate was used to induce DNA precipitation20. The yield of DNA pellet improved after incubating overnight at –20oC. The DNA pellet was dissolved in sterile distilled water and not in Tris–EDTA buffer since the same can interfere in PCR amplification, as EDTA was found to effectively reduce the magnesium concentration21.

In our experiment we used both Taq polymerase and a DNA polymerase from Thermus brockianus (Dynazyme II) for PCR amplification. Both the polymerases amplified the phytoplasma DNA (Figure 1). In the subsequent studies only Taq polymerase was used and a 558 bp fragment of DNA was amplified from both the Kerala and Karnataka sandal spike populations (Figure 2). The last extension step was prolonged to 5 min to ensure that all the PCR products are of full length22.

The restriction enzyme AluI has the recognition sequence 5 -AG/CT-3 . The restriction enzyme was incubated with the PCR product overnight at 37oC rather than for 2 h to increase the efficiency during further detection by electrophoresis. As the PCR product in the study was 558 bp, TAE buffer and 1.5% agarose gel were used since the gel had an effective resolution in the range 3–0.2 kb (ref. 23). TBE buffer and 3% agarose gel were employed to visualize smaller DNA fragments less than 0.2 kb, while TBE buffer provided good resolution than TAE buffer24.

The RFLP analysis with AluI restriction endonuclease confirms that sandal phytoplasma could be attributed to the first group of phytoplasmas as proposed by Ahrens and Seemuller9. Different workers have identified phytoplasmas of the first group associated with diseases in lettuce25, declining apricot26, pear decline27, dieback in papaya28, periwinkle29, corn poppy30, etc. The same technique could be used to detect and classify phytoplasmas affecting different plants in India.


  1. Ghosh, S. K., Balasundaran, M. and Ali, M. I. M., in Plant Diseases of International Importance (eds Mukhopadhyay, A. N., Kumar, J. and Chaube, H. S.), Prentice Hall, USA, 1992, vol. IV, pp. 296–310.
  2. Varma, A., Chenulu, V. V., Raychaudhuri, S. P., Prakash, N. and Rao, P. S., Indian Phytopathol., 1969, 22, 289–294.
  3. Hull, R., Horne, R. W. and Nayar, R. M., Nature, 1969, 224, 1121–1122.
  4. Ghosh, S. K., Balasundaran, M. and Ali, M. I. M., in Studies on the Spike Disease of Sandal, KFRI Research Report no. 37, KFRI, Kerala, 1985, pp. 8–31.
  5. Hiruki, C., Giannotti, J. and Dijkstra, J., Neth. J. Plant Pathol., 1974, 80, 145–153.
  6. Parthasarathy, K., Gupta, S. K. and Rao, P. S., Proc. Indian Acad. Sci. Sect. B, 1966, 64, 152–156.
  7. Thomas, S. and Balasundaran, M., Curr. Sci., 1998, 74, 989–993.
  8. McCoy, R. E., Caudwell, A., Dhang, C. J., Chiykowski, L. N., Cousin, M. T., Dale, J. L., de Leeuw, G. T. N., Golino, D. A.,
  9. Hackett, K. J., Kirckpatrick, B. C., Marwitz, R., Petzold, H., Sinha, R. C., Suguira, M., Whitcomb, R. F., Yang, I. L., Zhu,
    B. M. and Seemuller, E., in The Mycoplasmas, vol. 5, Spiroplasmas, Acholeplasmas and Mycoplasmas of Plants and Arthropods (eds Whitcomb, R. F. and Tully, J. G.), Academic Press, USA, 1989, pp. 545–640.
  10. Ahrens, U. and Seemuller, E., Phytopathol., 1992, 82, 828–832.
  11. Gundersen, D. E., Lee, I. M., Renher, S. A., Davis, R. E. and Kingsbury, D. T., J. Bacteriol., 1994, 176, 5244–5254.
  12. Seemuller, E., Schneider, B., Maurer, R., Ahrens, U., Daire, X., Kison, H., Lorenz, K.H., Firrao, G., Avinent, L., Sears, B. B. and Stackebrandt, E., Int. J. Syst. Bacteriol., 1994, 44, 440–446.
  13. Namba, S., Oyaizu, H., Kato, S., Iwanami, S. and Tsuchizaki, T., Int. J. Syst. Bacteriol., 1993, 43, 461–467.
  14. Schneider, B., Cousin, M. T., Klinkong, S. and Seemuller, E.,
    Z. Pflanzenkrankh. Pflanzenschutz, 1995, 102, 225–232.
  15. Sinclair, W. A., Griffiths, H. M. and Davis, R. E., Plant Dis., 1996, 80, 468–475.
  16. Doyle, J. J. and Doyle, J. L., Focus, 1990, 12, 13–15.
  17. Lim, P. O. and Sears, B. B., J. Bacteriol., 1989, 171, 5901–5906.
  18. Schneider, B., Marcone, C., Kampmann, M., Ragozzino, A., Lederer, W., Cousin, M. T. and Seemuller, E., Eur. J. Plant Pathol., 1997, 103, 675–686.
  19. Woese, C. R., Maniloff, J. and Zablen, L. B., Proc. Natl. Acad. Sci. USA, 1980, 77, 494–498.
  20. Moore, D., in Current Protocols in Molecular Biology (eds Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K.), John Wiley and Sons, USA, 1996, vol. I, unit 2.1.
  21. Reichardt, M. and Rogers, S., in Current Protocols in Molecular Biology (eds Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K.), John Wiley and Sons, USA, 1996, vol. I, unit 2.3.
  22. Ruiz, P., Haasner, D. and Wiles, M. V., in Immunology Methods Manual – The Comprehensive Sourcebook of Techniques (ed. Lefkovits, I.), Academic Press, USA, 1997, vol. I, pp. 287–304.
  23. Kramer, M. F. and Coen, D. M., in Current Protocols in
    Molecular Biology
    (eds Ausubel, F. M., Brent, R., Kingston,
    R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K.), John Wiley and Sons, USA, 1996, vol. 2, unit 15.
  24. Voytas, D., in Current Protocols in Molecular Biology (eds Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K.), John Wiley and Sons, USA, 1996, vol. I, unit 2.5.
  25. Martin, R., in Gel Electrophoresis: Nucleic Acids, Bios Scientific Publishers, UK, 1996, pp. 75–99.
  26. Marcone, C., Ragozzino, A., Firrao, G. and Locci, R., Petria, 1995, 5, 111–118.
  27. Marcone, C., Ragozzino, A., Firrao, G. and Locci, R., Petria, 1995, 5, 143–152.
  28. Pollini, C. P., Giunchedi, L., Seemuller. E. and Lorenz, K. H.,
    J. Phytopathol., 1994, 142, 115–121.
  29. Gibb, K. S., Persley, D. M., Schneider, B. and Thomas, J. E., Plant Dis., 1996, 80, 174–178.
  30. Marcone, C. and Ragozzino, A., Riv. Patol. Veg., 1995, 5, 49–59.
  31. Marcone, C., Ragozzino, A. and Seemuller, E., J. Plant Dis. Prot., 1996, 103, 82–84.

ACKNOWLEDGEMENTS.  The authors thank the Department of Biotechnology, Govt. of India for financial support. S.T. acknowledges CSIR, Govt. of India for a senior research fellowship.

Received 30 December 1998; revised accepted 22 March 1999