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Polymerase Chain Reaction-Based Detection of Spiroplasma citri Associated with Citrus Stubborn Disease

Polymerase chain reaction (PCR)-based detection of citrus stubborn disease was improved using primers based on sequences of the P89 putative adhesin gene and the P58 putative adhesin multigene of Spiroplasma citri. Real-time PCR also was developed with detection limits estimated to be between 10^--4 and 10^--4 ng by serial dilution of a recombinant S. citri plasmid into DNA extracts from healthy Madam Vinous sweet orange. PCR for the detection of S. citri by these new primers was validated by comparing culturing of the pathogen, the traditional method of diagnosis, with PCR assays from samples taken from two citrus plots in Kern County, CA. Fruit columella was collected from 384 and 377 individual trees in each of two fields, respectively; one portion was used for culturing and the other for DNA extraction and PCR. PCR results matched those of culturing 85 to 100% of the time depending on the primers used. More importantly, PCR detected S. citri from culture-negative trees in 5 to 15% of the cases, suggesting that PCR performed as well or better than culturing for detection of S. citri in field samples. Real-time PCR proved to be the best method for detection. Differential reaction of the samples to the P58 primer pairs suggested that two populations of S. citri occur in historical and present-day field isolates. Citrus stubborn disease incidence was estimated to be 58.3 and 3.7% in the two orchards. The results presented here support the use of PCR for reliable detection of S. citri in field trees.