Submitted to: Plant Disease
Publication Type: Peer reviewed journal
Publication Acceptance Date: 7/26/2014
Publication Date: 1/5/2015
Citation: Wang, X., Doddapaneni, H., Chen, J., Yokomi, R.K. 2015. Improved real-time PCR diagnosis of citrus stubborn disease by targeting prophage genes of Spiroplasma citri. Plant Disease. 99:149-154. Interpretive Summary: Citrus stubborn disease (CSD) is caused by Spiroplasma citri, a microorganism that grows in sap-conducting tissue of infected plants. A wide variety of plant hosts are susceptible to infection after leafhopper vectors harboring the pathogen feed. Affected plants include many weeds and ornamental species and crops such as potatoes, carrots, horseradish, sesame and citrus. Pathogen concentration in citrus varies widely with season and is generally low and unevenly distributed, making detection difficult. Presently, detection relies on a diagnostic procedure called polymerase chain reaction (PCR). Current genetic markers for PCR detection of S. citri are present in single or low copy numbers in the chromosome of the pathogen. The purpose of this research was to improve S. citri detection by selecting genetic markers that are present in multiple copies in the pathogen’s chromosome. A search of S. citri genome sequences revealed presence of prophage virus genomes embedded within the chromosome of S. citri. Two prophage sequences, Php-orf1 and Php-orf3, were selected and used as markers in quantitative PCR (qPCR) assays for S. citri. The prophage markers successfully identified all 18 diverse S. citri cultures isolated from different hosts and locations. Results indicated PCR sensitivity was increased 1,000- to nearly 100,000-fold for Php-orf3 and Php-orf1, respectively, over single copy marker genes. The prophage marker sets also were evaluated for S. citri detection in 252 field trees from two citrus orchards in central California known to be infected by S. citri. The prophage markers detected all CSD-infected trees identified by single copy gene markers and, more importantly, detected 10 and 6 additional infected trees, respectively. PCR assays with prophage markers proved much more sensitive and decreased false negatives. The new detection assays are now being used to determine epidemiology of CSD and to improve CSD management.
Technical Abstract: Spiroplasma citri is a phloem-limited bacterium causing citrus stubborn disease (CSD). Isolation and culturing of S. citri is difficult and time consuming. Current detection methods use polymerase chain reaction (PCR) assays with primers developed from sequences of S. citri house-keeping genes. In citrus, S. citri is typically low in titer and unevenly distributed, hence, detection is challenging. Recent genome sequencing of S. citri revealed that the bacterium harbors multiple copies of prophage genes. Targeting multicopy prophage genes could lead to more sensitive detection. Two primer sets, Php-orf1 and Php-orf3, were developed from conserved prophage sequences in the S. citri genome. The primers were used in SYBR Green-based quantitative PCR(qPCR) assays to evaluate detection sensitivity with 18 S. citri cultures isolated from different hosts and locations. Prophage primer set Php-orf1 increased detection sensitivity by approximately 4.91 and 3.65 log units compared with housekeeping gene primers for spiralin and P58 putative adhesin gene, respectively. Similarly, Php-orf3 primer increased detection sensitivity by 3.02 and 1.76 log units over assays using the same primers, respectively. Both prophage primer sets were validated for sensitive qPCR detection with 252 field samples from two citrus orchards in California’s San Joaquin Valley collected from 2007 to 2011. The data presented indicated use of PCR primers of S. citri prophage sequences resulted in more sensitive detection of the pathogen. The enhanced sensitivity is the result of higher copy number of prophage genes found in the S. citri genome and, thus, can be used for CSD diagnosis from field samples.