|Yokomi, Raymond - Ray|
Submitted to: American Phytopathological Society Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 6/12/2007
Publication Date: 7/1/2007
Citation: Yokomi, R.K., Mello, A.F., Saponari, M., Fletcher, J. 2007. PCR-based Detection of Spiroplasma citri Associated with Citrus Stubborn Disease. Phytopathology. 97:S127.
Technical Abstract: PCR detection of Spiroplasma citri, the causal agent of citrus stubborn disease, was improved using primers based on sequences of the P89 adhesin gene and the P58 putative adhesin multigene of S. citri. PCR was compared with isolation by culturing for detection of S. citri in two 20 A citrus orchards in Kern Co., Calif. Three fruit per tree were harvested from individual trees regardless of it’s symptomotology from 6 replicate blocks of 64 (8 x 8) trees each at different locations per field. Columella was excised from each fruit and the peduncle portion used for culturing while the remaining portion was subjected to DNA extraction and PCR. Of the 760 trees tested, PCR matched culture results 93.8% of the time; while 5% of the samples were PCR positive and culture negative and 1.2% were culture positive and PCR negative. Real time PCR was also developed for S. citri detection using two different P58 sequence primer sets. Differential reaction to these primers suggested at least two genotypes of S. citri exist in the plots. The P89 and P58 primer sets were equivalent and both were far more sensitive than spiralin gene primers for S. citri detection. These data indicated an estimated disease incidence of 60% and 2.4% in the two orchards and support the use of PCR for reliable detection of S. citri in field trees. Effective detection of the stubborn pathogen from field trees by PCR facilitates epidemiology studies.