|Yokomi, Raymond - Ray|
Submitted to: Journal of Plant Pathology
Publication Type: Abstract Only
Publication Acceptance Date: 11/30/2007
Publication Date: 8/24/2008
Citation: Yokomi, R.K., Mello,, A.F., Fletcher,, J. 2008. Citrus stubborn disease incidence determined by quantitative real time PCR. Journal of Plant Pathology 90 (2, Suppl.) S2.465.
Technical Abstract: Quantitative real-time (q) PCR was developed for detection of Spiroplasma citri, the causal agent of citrus stubborn disease (CSD), using the DNA binding fluorophore SYBR Green I. The primer pair, P58-3f/4r, developed based on sequences from the P58 putative adhesin multigene of the pathogen resulted in an amplicon of an estimated size of 119 bp from S. citri cultures and from DNA extracts of various tissues taken from field- and greenhouse-grown citrus trees. Using qPCR, estimates of CSD incidence in two 12 ha navel orange groves in Kern County, CA, determined by sampling individual trees in six different groups of 8x8 trees per grove were 60% and 4%. All qPCR positives were confirmed by cultivation of S. citri in LD8 liquid medium. A second primer pair, P58-1f/2r, yielded an amplicon of approximately 86 bp and reacted with a smaller group of S. citri strains from lab and greenhouse cultures as well as from infected field trees, providing support for the presence of two populations of S. citri in California. The qPCR technology is being applied in an assessment of within-tree pathogen distribution as well as in a study of CSD incidence and diversity in central California.