|Mello,, Alexandre - OKLAHOMA ST.UV, STILLWATE|
|Fletcher,, Jacqueline - OKLAHOMA ST.UV, STILLWATE|
|Saponari, Maria - INSTITUTE DI VIROLOGIA|
Submitted to: International Organization of Citrus Virologists Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: August 20, 2007
Publication Date: October 17, 2007
Citation: Yokomi, R.K., Mello,, A.F., Fletcher,, J., Saponari, M. 2007. Quantitative Detection of Spiroplasma Citri by Real Time PCR. In: Proceedings of the 17th Conference International Organization of Citrus Virologists, October 22-26, 2007, Adana, Turkey. p. 172. Technical Abstract: There is a need to develop an accurate and rapid method to detect Spiroplasma citri, the causal agent of citrus stubborn disease for use in epidemiology studies. Quantitative real-time PCR was developed for detection of S. citri. Two sets of primers based on sequences from the P58 putative adhesin multigene of S. citri resulted in amplicons of estimated sizes of 86 (P 58 1f/2r) and 119 bp.(P 58 3f/4r) The DNA binding fluorophore SYBR Green I was used for the real-time assay. The assay detected S. citri from cells in culture and in DNA extracts from columella and leaf petioles from stubborn-infected trees from the greenhouse and field. The assay was estimated to be sensitive to a level of less than 10-6 ng of S. citri DNA using the broad spectrum primer P58-3f/4. Primer P58 1f/2r, however, reacted with a smaller population of S. citri strains. The assay successfully detected S. citri in two field plots in Kern County and identified two genetic populations in the field as well as from stubborn strains collected in the 1970’s and maintained continuously in planta. Real time PCR should now provide a reliable new tool to assess stubborn disease incidence. Stubborn is a phloem limited prokaryote like huanglongbing (HLB). The two diseases have similar symptomotology, however, HLB does not occur in California. Proactive surveys are underway in California for HLB. Therefore, the S. citri test can be used with the HLB test to sort out infections.