Muhammad Nauman, Ummad U.D. Umar, Syed A.H. Naqvi, Ateeq U. Rehman, Muhammad T. Malik, Muhammad Shahid, Muhammad Akbar, Muhammad Umair


Citrus greening/ yellow shoot disease/ HLB, caused by Candidatus liberibacter asiaticus, showing mosaic/ mottling pattern on leaves, stunting of plants, de-shape, pre-mature fruit drop and yellowing of reticulate venation as characteristic symptoms, is the most concerned disease prevailing in citrus groves of Punjab, Pakistan. For the detection of pathogen and downstream studies, a high-quality DNA is required. In citrus, due to the variety of species, different age groups in plants, thick waxy cuticle of leaves, high production of phenolics, polysaccharides and other compounds, it is very difficult to extract good quality DNA from leaves and especially from main midrib where the fastidious bacterium is residing. CTAB and SDS are two devised methods for the extraction of total genomic DNA from citrus leaves while the current research suggests some modified protocols for the detection of DNA from infected/ healthy samples. 100mg leaf midrib sample was crushed in liquid nitrogen, homogenized in 500µl CTAB, incubated at 60°C in water bath for 45 minutes, centrifuged at 12000rpm for 5 minutes, supernatant was transferred to new tube and 5µl RNase-A was added, incubated at 37°C for 20 minutes. Equal volume of chloroform/ isoamyl alcohol (24:1) was added, vortexed for 5 minutes, centrifuged for 2 minutes at 12000rpm for phase separation and upper phase was transferred to new tube where 500µl of chilling isopropanol was added, kept at -20°C for 15 minutes, centrifuged at 12000rpm, pallet was washed twice with 70% ethanol. CTAB method was modified by increasing the incubation time i.e., 45 minutes for cell lysis in water bath for determination the variation in quality of DNA and the amount of beta mercaptethanol was also doubled from the normal one. Modified protocols have been proved excellent for the extortion of total genomic DNA from citrus. In SDS, addition of TE buffer, RNAase, Isoamylalcohol: Chloroform (24:1) along with 20% SDS and 2M Sodium Acetate gave high quality DNA from citrus leaves. Hence, DNA was extracted by four different ways but the modified CTAB and SDS methods gave improved quality DNA as confirmed by its quantification through nanophotometer. The DNA was quantified by nanophotometer and presence was observed on gel documentation apparatus. Hence, DNA was found suitable for PCR and RFLP analysis and long-term storage on -80 ⁰C.


Greening, Extraction Protocol, Fastidious bacteria, Molecular plant pathology.

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Bové, J., M. E. Dwiastuti, A. Triviratno, A. Supriyanto, E. Nasli, P. Becu and M. Garnier. 2000. Incidence of huanglongbing and citrus rehabilitation in North Bali, Indonesia. International Organization of Citrus Virologists Conference Proceedings (1957-2010).

Bové, J. M. 2006. Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. Journal of plant pathology, 7-37.

Chung, K.R. and R. H. Brlansky. 2006. Citrus Diseases Exotic to Florida: Citrus Tristeza Virus, Stem Pitting. Plant Pathology Department, Florida Cooperative Extension Service, Institute of Food, pp. 210.

Coletta-Filho, H. D., M. L. P. N. Targon, M. A. Takita, J. D. De Negri, J. Pompeu, M. A. Machado, A. M. do Amaral and G. W. Muller. 2004. First Report of the Causal Agent of Huanglongbing (“Candidatus liberibacter asiaticus”) in Brazil. Plant Disease, 88: 1382-1382.

de la Cruz, M., F. Ramirez and H. Hernandez. 1997. Plant Molecular Biology Reporter, 15: 319-325.

Dellaporta, S. L., J. Wood and J. B. Hicks. 1983. A plant DNA minipreparation: Version II. Plant Molecular Biology Reporter, 1: 19-21.

Deng, X., Y. Lan, T. Hong and J. Chen. 2016. Citrus greening detection using visible spectrum imaging and C-SVC. Computers and Electronics in Agriculture, 130: 177-183.

Eveland, W. G. and K. E. Brown. 2019. Method of Treating Citrus Greening. Google Patents.

Fang, G., S. Hammar and R. Grumet. 1992. A quick and inexpensive method for removing polysaccharides from plant genomic DNA. Biotechniques, 13: 52-54.

Fraser, L. R. and D. Singh. 1968. Citrus dieback in India—the contribution of greening virus. International Organization of Citrus Virologists Conference Proceedings (1957-2010).

Fujikawa, T. and T. Iwanami. 2012. Sensitive and robust detection of citrus greening (huanglongbing) bacterium “Candidatus liberibacter asiaticus” by DNA amplification with new 16S rDNA-specific primers. Molecular and Cellular Probes, 26: 194-197.

Garnier, M. 1983. Transmission of the Organism Associated with Citrus Greening Disease from Sweet Orange to Periwinkle by Dodder. Phytopathology, 73: 1358.

Garnier, M. and J. M. Bové. 1993. Citrus greening disease and the greening bacterium. International Organization of Citrus Virologists Conference Proceedings (1957-2010).

Gottwald, T. R. 1989. Preliminary Analysis of Citrus Greening (Huanglungbin) Epidemics in the People's Republic of China and French Reunion Island. Phytopathology, 79: 687.

Hong, Y., Y. Luo, J. Yi, L. He, L. Dai and T. Yi. 2019. Screening nested-PCR primer for ‘Candidatus Liberibacter asiaticus’ associated with citrus Huanglongbing and application in Hunan, China. PLOS ONE, 14: e0212020.

Jagoueix, S., J. M. Bove and M. Garnier. 1994. The Phloem-Limited Bacterium of Greening Disease of Citrus Is a Member of the Subdivision of the Proteobacteria. International Journal of Systematic Bacteriology, 44: 379-386.

Jagoueix, S., J. M. Bové and M. Garnier. 1996. PCR detection of the two Candidatus liberobacter species associated with greening disease of citrus. Molecular and Cellular Probes, 10: 43-50.

Manjunath, K. L., S. E. Halbert, C. Ramadugu, S. Webb and R. F. Lee. 2008. Detection of ‘Candidatus liberibacter asiaticus’ in Diaphorina citri and Its Importance in the Management of Citrus Huanglongbing in Florida. Phytopathology, 98: 387-396.

Möller, E. M., G. Bahnweg, H. Sandermann and H. H. Geiger. 1992. A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissues. Nucleic Acids Research, 20: 6115-6116.

Planet, P., S. Jagoueix, J. M. Bov and M. Garnier. 1995. Detection and characterization of the African citrus greening Liberobacter by amplification, cloning, and sequencing of the rplKAJL-rpoBC operon. Current Microbiology, 30: 137-141.

Porebski, S., L. G. Bailey and B. R. Baum. 1997. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Molecular Biology Reporter, 15: 8-15.

Saghai-Maroof, M. A., K. M. Soliman, R. A. Jorgensen and R. W. Allard. 1984. Ribosomal DNA spacer-length polymorphisms in barley: mendelian inheritance, chromosomal location, and population dynamics. Proceedings of the National Academy of Sciences, 81: 8014-8018.

Schlink, K. and R. Reski. 2002. Preparing high-quality DNA from moss (Physcomitrella patens). Plant Molecular Biology Reporter, 20: 423-423.

Spreen, T. H., J.-P. Baldwin and S. H. Futch. 2014. An Economic Assessment of the Impact of Huanglongbing on Citrus Tree Plantings in Florida. HortScience, 49: 1052-1055.

Teixeira, D. d. C., C. Saillard, S. Eveillard, J. L. Danet, P. I. d. Costa, A. J. Ayres and J. Bové. 2005. ‘Candidatus liberibacter americanus’, associated with citrus huanglongbing (greening disease) in São Paulo State, Brazil. International Journal of Systematic and Evolutionary Microbiology, 55: 1857-1862.

Yaqub, M. S. 2017. Molecular detection of Candidatus liberibacter asiaticus, the causal organism of huanglongbing (citrus greening) in faisalabad, pakistan for huanglongbing management. Pakistan Journal of Agricultural Sciences, 54: 21-26.



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Pakistan Journal of Phytopathology
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