IJBTT

Distinguishing Between Two Closely Related Strains of Xylella fastidiosa

	International Journal of Biotech Trends and Technology (IJBTT)
	© 2014 by IJBTT Journal
	Volume - 4 Issue - 4
	Year of Publication : 2014
	Authors : Arinder K. Arora, Ravi Durvasula, Thomas A. Miller

Citation

Arinder K. Arora, Ravi Durvasula, Thomas A. Miller "Distinguishing Between Two Closely Related Strains of Xylella fastidiosa", International Journal of Biotech Trends and Technology (IJBTT), V4(4):8-13 Oct - Dec 2014, Published by Seventh Sense Research Group.

Abstract

A real time PCR assay (rt-PCR), using Taqman minor groove binder (MGB) probes was developed to quantify and differentiate two strains of Xylella fastidiosa, Pierce’s Disease (PD) strain and Almond Leaf Scorch (ALS) strain. Two isolates, Temecula and Dixon, were used as references for the PD strain and the ALS strain, respectively. Primers and probes based on the gyraseB sequence were able to distinguish between both the isolates, and DNA concentrations as low as 1.01×10-5 ng/µl (3 copy number/µl) were detected. MGB probes were also able to distinguish between the Temecula and Dixon isolate DNA from a mixture of the two DNA’s. Whereas the Temecula specific MGB probe showed 106–fold resolution, the Dixon specific probe showed only 10-fold resolution. We have developed a reliable, sensitive and specific rt-PCR assay using MGB probes to quantify and distinguish DNA of the two strains of X. fastidiosa.

References

[1] J. M. Wells, B. C. Raju, H. Y. Hung, W. G. Weisburg, L. Mandelco-Paul, and D. J. Brenner, "Xylella fastidiosa gen. nov., sp. Nov.: Gram-negative, xylem-limited fastidious plant bacteria related to Xanthomonas spp.," Int. J. Syst. Bacteriol., vol. 37, pp. 136-143, 1987.
[2] D. L. Hopkins, ”Xylella fastidiosa: xylem-limited bacterial pathogen of plants," Annu. Rev. Phythopathol., vol. 27, pp. 271-90, 1989.
[3] M. J. Blua,, P. A. Phillips and R. A. Reda, "A new sharpshooter threatens both crops and ornamentals," Calif. Agric., vol. 53, pp. 22-25, 1999.
[4] D. M. Takiya, S. H. Mckamey, and R. R. Cavichiloi, "Validity of Homalodisca and of H. vitripennis as the name for glassy-winged sharpshooter (Hemiptera: Cicadellidae: Cicadellinae)," Annals. of the Entomological Society of America, vol. 99, pp. 648-655, 2006.
[5] M. R. Pooler, and J. S. Hartung, " Genetic relationships among strains of Xylella fastidiosa from RAPD-PCR data," Curr. Microbiol., vol. 31, pp. 134–137, 1995.
[6] M. Hendson, A. Purcell, D. Chen, C. Smart, M. Guilhabert, and K. Kirkpatrick, "Genetic diversity of Pierce’s disease strains and other pathotypes of Xylella fastidiosa," Appl. Environ. Microbiol., vol. 67, pp. 895–903, 2001.
[7] J. Chen, J. S. Hartung, C. Chang, and A. Vidaver, "An evolutionary perspective of Pierce’s Disease of grapevine, citrus variegated chlorosis, and mulberry leaf scorch diseases," Curr. Microbiol., vol. 45, pp. 423–428, 2002.
[8] L. L. R. Marques, H. Ceri, G. P. Manfio, D. M. Reid, and M. E. Olson, "Characterization of biofilm formation by Xylella fastidiosa in vitro," Plant Dis., vol. 86, pp. 633–638, 2002.
[9] J. L. M. Rodrigues, M. E. Silva-Stenico, J. E. Gomes, J. R. S. Lopes and S. M. Tsai, "Detection and diversity assessment of Xylella fastidiosa in field-collected plant and insect samples by using 16S rRNA and gyrB sequences," Appl. Environ. Microb., vol.. 69, pp. 4249–4255, 2003.
[10] N. W. Schaad, E. Postnikova, G. Lacy, M. Fatmi, and C. J. Chang, " Xylella fastidiosa subspecies: X. fastidiosa subsp piercei, subsp. Nov., X. fastidiosa subsp. Multiplex subsp. Nov., and X. fastidiosa subsp. Pauca subsp. Nov.,`" Syst. Appl. Microbiol., vol. 27, pp. 290–300, 2004.
[11] E. L. Schuenzel, M. Scally, R. Stouthamer, and L. Nunney, "A multigene phylogenetic study of clonal diversity and divergence in North American strains of the plant pathogen Xylella fastidiosa, " Appl. Environ. Microbiol. vol. 71, pp. 3832-3839, 2005.
[12] B. Bextine and B. Child, " Xylella fastidiosa genotype differentiation by SYBR Green-based QRT-PCR," FEMS Microbiol. Lett., vol. 276, pp. 48–54, 2007.
[13] N. W. Schaad, J. B Jones, and W. Chun, W., Laboratory Guide for Identification of Plant Pathogenic Bacteria 3rd ed, APS press, Saint Paul, MN, 2001.
[14] R. Hernandez-Martinez, H. S. Costa, C. K. Dumenyo and D. A. Cooksey, " Differentiation of Strains of Xylella fastidiosa Infecting Grape, Almonds, and Oleander Using a Multiprimer PCR Assay," Plant Dis., vol. 90, pp. 1382-1388, 2006.
[15] N. W. Schaad, D. Opgenorth, and P. Gaush, "Real-time polymerase chain reaction for one-hour on-site diagnosis of Pierce’s disease of grape in early season asymptomatic vines," Phytopathology, vol. 92, pp. 721-728, 2002.
[16] R.P.P. Almeida, and A. H. Purcell, "Biological traits of Xylella fastidiosa strains from grapes and almonds," Appl. Environ. Microbiol., vol. 69, pp. 7447-7452, 2003
[17] R.P.P. Almeida, and A. H. Purcell H, "Homalodisca oagulate (Hemiptera: Cicadellidae) transmission of Xylella fastidiosa to almond," Plant Dis., vol. 87, pp. 1255-1259, 2003.
[18] I. Afonina, M. Zivarts, I. Kutyavin, E. Lukhtanov, H. Gamper, and R. B. Meyer, "Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder," Nucleic Acids Res., vol. 25, pp. 2657-2660, 1997.
[19] I. V. Kutyavin, I. A. Afonina, A. Mills, V. V. Gorn, E. A. lukhtanov, E. S. Belousov, M. J. Singer, D. K. Walburger, S. G. Lokhov, A. A. Gall, R. Dempcy, M. W. Reed, R. B. Meyer, and J. Hedgpeth, "3?-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures," Nucleic Acids Res., vol. 28, pp. 655–661, 2003.
[20] K. L. Newman, R. P. P. Almeida, A. H. Purcell, and S. E. Lindow, " Use of a green fluorescent strain for analysis of Xylella fastidiosa colonization of Vitis vinifera," Appl. Environ. Microbiol, vol. 69, pp. 7319-7327, 2003.
[21] J. Chen, R. Groves, E. L. Civerolo, M. Viveros, M. Freeman and Y. Zheng," Two Xylella fastidiosa genotypes associated with almond leaf scorch disease on the same location in California," Phytopathology, vol. 95, pp. 708-714, 2005.

Keywords
Real time-PCR, Xylella fastidiosa, strains, PD, ALS, Temecula, Dixon.