Submitted to: Phytopathology
Publication Type: Abstract Only
Publication Acceptance Date: 5/10/2013
Publication Date: 6/30/2013
Citation: Wang, X., Yokomi, R.K., Chen, J. 2013. Sensitive detection of Spiroplasma citri by targeting prophage sequences. Phytopathology. 103:S2.157.
Technical Abstract: Spiroplasma citri is a phloem limited and nutritionally fastidious wall-less bacterium causing citrus stubborn disease (CSD) in California. An important step in CSD management is early detection of S. citri. Because isolation of S. citri is technically demanding and time consuming, current detection protocols are exclusively PCR-based. Current PCR primers were developed from sequences of house-keeping genes such as 16S rRNA, spiralin and putative adhesin (P58), which have low copy numbers in the bacterial genome. Recent advance in genome sequencing of S. citri revealed that the bacterium harbors multiple copies of prophage genes. The multi-copy DNAs provides higher template concentration, leading to more sensitive PCR detection. In this study, two primer sets (Php-orf1 and Php-orf3) were developed from prophage sequences in the S. citri genome. SYBR Green-based real-time PCR was performed to evaluate detection sensitivity with 18 S. citri cultures isolated from different hosts including citrus, peach, horseradish and carrot. Compared with the primer sets based on spiralin and P58 gene, primer set Php-orf1 reduced Ct values by 3.02±0.62 and 1.76±0.51, respectively. Similarly, primer set Php-orf3 reduced Ct value by 4.91±0.49 and 3.65±0.62, respectively. With >250 field samples collected in two citrus orchards in the central valley of California from 2007 to 2011, an over three log increase of detection sensitivity was also observed. In conclusion, results from this study showed PCR targeting prophage sequences was a highly sensitive technique for detection of S. citri.