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Title: CHARACTERIZATION OF A NOVEL ADHESIN GENE AND DESIGN OF A REAL-TIME POLYMERASE CHAIN REACTION FOR RAPID, SENSITIVE, AND SPECIFIC DETECTION OF SPIROPLASMA KUNKELII

Author
item WEI, WEI - CHINA
item OPGENORTH, DAN - DEPT OF FOOD AND AGRI CA
item Davis, Robert
item CHANG, CHUNG-JAN - UNIV OF GA GRIFFIN GA
item SUMMERS, CHARLES - UNIV OF CA DAVIS CA
item Zhao, Yan

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/11/2006
Publication Date: 5/11/2006
Citation: Wei, W., Opgenorth, D., Davis, R.E., Chang, C., Zhao, Y. 2006. Characterization of a novel adhesin gene and design of a real-time polymerase chain reaction for rapid, sensitive, and specific detection of spiroplasma kunkelii. Plant Disease. 90:1233-1238.

Interpretive Summary: Spiroplasma kunkelii is a small bacterium without a cell wall. It causes a serious corn disease known as corn stunt, which is widespread in the Americas and has been a costly problem to corn growers, dairymen, and ethanol producers. The bacterium lives in nutrient-conducting cells of affected plants and is spread by a group of insects called leafhoppers. Early detection of the bacterium in disease-spreading leafhoppers and in infected plants is a key to disease forecasting and to outbreak management. In the present study, a unique genetic unit within the DNA of the bacterium was identified and the new genetic information was used to develop a diagnostic tool for rapid, sensitive, and reliable detection of the bacterium. The diagnostic tool developed in this study was able to detect extremely low levels of the bacterium and was successfully applied to quick detection of the bacterium in field plants and leafhoppers. This report will be of interest to corn growers, extension personnel, and scientists who are concerned with corn diseases.

Technical Abstract: 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 till close to flowering time, early detection of the pathogen in disease-transmitting leafhoppers and in symptom-less foliar tissues of host plants is critical to disease forecasting and to 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 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 over that of conventional PCR. The assay was found to be highly specific to S. kunkelii as it did not cross-react even with 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.