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United States Department of Agriculture

Agricultural Research Service

Research Project: CHARACTERIZATION & EPIDEMIOLOGY OF CITRUS TRISTEZA VIRUS & OTHER INVASIVE & EMERGING GRAFT-TRANSMISSIBLE DISEASES OF CITRUS IN CALIFORNIA Title: PCR-based Detection of Spiroplasma Citri Associated with Citrus Stubborn Disease

Authors
item Yokomi, Raymond
item Mello, Alexandre - OKLAHOMA ST UV-STILWATER
item Saponari, Maria - INST DI VIROLOGIA,ITALY
item Fletcher, Jaqueline - OKLAHOMA ST UV-STILWATER

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 17, 2007
Publication Date: February 1, 2008
Citation: Yokomi, R.K., Mello, A., Saponari, M., Fletcher, J. 2008. PCR-based detection of spiroplasma citri associated with citrus stubborn disease. Plant Disease. 92:253-260.

Interpretive Summary: Polymerase chain reaction (PCR) detection of Spiroplasma citri, the citrus stubborn disease agent, was improved by designing new primers based on different gene sequences of the pathogen. Our new PCR procedure was compared to culturing of the bacterial disease agent and was found to be more reliable, easier to do, and much cheaper than culturing. The use of our PCR method was validated for stubborn detection by sampling and testing citrus trees in two 20 A citrus plots in northeastern Kern Co. in central California. Of the 760 trees tested, PCR matched culture results 93.8% of the time. Of the 6.2% mismatches, 5% were PCR positive and culture negative; whereas 1.2% were PCR negative and culture positive. This validation now supports the use of PCR for the identification of the pathogen in field trees. Quantitative, real time PCR was also developed for S. citri detection using two different P58 primer sets. These primers identified two different S. citri strains in the citrus plots. Research is continuing to determine if these strains have a different symptom phenotype or vector transmissibility.

Technical Abstract: Improvements in polymerase chain reaction (PCR) detection of Spiroplasma citri, the causal agent of citrus stubborn disease, made PCR more reliable than culturing for S. citri detection. Primer sequences from the P89 putative adhesin gene, which is present on a plasmid as well as in the S. citri genome, and the P58 putative adhesin multigene were found to be much more sensitive than spiralin gene primers. PCR was compared to isolations and culturing for stubborn detection in two 20 A citrus plots in northeastern Kern Co. Of the 761 trees tested, PCR matched culture results 93.8% of the time. This validation now supports the use of PCR for screening S. citri infection with subsequent confirmation by culturing and dark field microscopy. Quantitative, real time PCR was also developed for S. citri detection using two different P58 primer sets. These primers identified two different S. citri strains in the citrus plots. Research is continuing to determine if these strains have a different symptom phenotype or vector transmissibility.

Last Modified: 10/23/2014
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