Characterization of a Novel Adhesin-like Gene and Design of a Real-Time PCR for Rapid, Sensitive, and Specific Detection of Spiroplasma kunkelii

Spiroplasma kunkelii, a cell wall-less bacterium, is the causal agent of corn stunt disease. The pathogen is restricted to phloem sieve cells of infected plants and is transmitted by phloem-feeding leafhoppers. Since symptoms of corn stunt disease may not appear until close to flowering time, early detection of the pathogen in disease-transmitting leafhoppers and in symptomless foliar tissues of host plants is critical to disease forecasting and outbreak management. In this study, a field-deployable real-time polymerase chain reaction (PCR) assay was developed for sensitive and specific detection of S. kunkelii. Nucleotide sequence from a previously unreported adhesin-like gene was used to design primers and a fluorogenic probe. The assay was able to detect the presence of S. kunkelii DNA as low as 5 fg, a sensitivity 100 times more than that of conventional PCR. The assay was found to be highly specific to S. kunkelii, as it did not cross-react with one of the most closely related plant pathogenic spiroplasma species, S. citri. The assay was successfully applied to rapid field detection of S. kunkelii in its plant host and insect vectors.