Abstract
Citrus greening disease caused by a fastidious bacterium is an important graft transmissible disease in commercial citrus in India and other parts of the world. Polymerase chain reaction (PCR) is a sensitive and convenient method for detection of greening bacterium. A non-phenol chloroform method of DNA extraction was evaluated for DNA quality and PCR based detection of greening bacterium. The method was comparable with a commercial DNA extraction kit (Qiagen) and better than a CTAB based DNA extraction method. To improve the reliability, three primer sets (primers A, B, and C yielding amplicons of 1160 bp, 703 bp and 451 bp, respectively) and two polymerase enzymes (Taq polymerase and Klen Taq polymerase) were evaluated. The primer set C provided better amplification when compared to primer sets A and B. Primer C in combination with Taq polymerase provided amplification band at a DNA template concentration of 100 pg but good amplification band was obtained at still lower DNA template concentration of 0.1 pg when Klen Taq polymerase was used. The standardized PCR protocol combining non-phenol chloroform method of DNA isolation, primer set C and Klen Taq polymerase enzyme was found very effective in detecting greening bacterium in citrus trees. The sequence of cloned amplicon from 16S ribosomal RNA gene had 89–100 % sequence identity with corresponding sequence of Candidatus Liberibacter asiaticus from China, Brazil, Japan and Pune isolate of India, C. Liberibacter americnus from Brazil and C. Liberibacter africanus from Africa.
Similar content being viewed by others
References
Varma A, Ahlawat YS, Chakraborty NK, Garnier M & Bove JM, In Proc 12th Conf IOCV, IOCV Riverside, California (1993) pp 280–285.
Ahlawat YS, Indian J Agril Sci, 67 (1997) 51.
Baranwal VK, Mazumder S, Singh J, Suryanarayan V, Ghosh DK & Ahlawat YS, Indian Phytopath, 57 (2004) 164.
Villechanoux S, Garnier M, Rennadin J & Bove JM, Curr Microbiol, 24 (1992) 89.
Hung TH, Wu ML & Su HJ, Ann Phytopathol Soc Jpn, 65 (1999) 140.
Jagouiex S, Bove JM & Garnier M, Mol Cell Pro, 10 (1996) 43.
Ahlawat YS, Baranwal VK, Thinley, Doe Doe & Mazumdar S, Plant Dis, 87 (2003) 448.
Hung, TH, Hung SC, Chen, CN, Hsu MH & Su HJ, Plant Pathol, 53 (2004) 96.
Li W, Hartung JS & Levy L. J Microbiol Meth, 2006.
Baranwal VK, Majumder S, Ahlawat YS & Singh R P, J Virol Meth, 112 (2003) 153.
Murray MG & Thompson WF, Nucl Acids Res, 8 (1980) 4321.
Sambrook J & Russell DW, Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, New York (2001)
Hocquellet A, Bove JM & Garnier M, In Proc 14th Conf IOCV, IOCV Riverside, California (2000) pp 363–368.
Harakava R, Morais LJ, Ochasou J, Manjuanth KL, Febres VJ, Lee, RF & Niblett, GL, In Proc 14th Conf IOCV, IOCV Riverside, California (2000) pp 195–199.
Hall TA, Nucl Acids Symp Ser, 41 (1999) 95.
Singh M & Singh RP, Can J Plant Pathol, 19 (1997) 149.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Gouda, K.A., Baranwal, V.K. & Ahlawat, Y.S. Simplified DNA Extraction and Improved PCR Based Detection of Greening Bacterium in Citrus. J. Plant Biochem. Biotechnol. 15, 117–121 (2006). https://doi.org/10.1007/BF03321914
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF03321914