Location: Crop Improvement and Protection ResearchTitle: Novel primers and PCR protocols for the specific detection and quantification of Sphingobium suberifaciens in situ Author
Submitted to: Molecular and Cellular Probes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/9/2014
Publication Date: 3/1/2014
Citation: Bull, C.T., Goldman, P.H., Martin, K.J. 2014. Novel primers and PCR protocols for the specific detection and quantification of Sphingobium suberifaciens in situ. Molecular and Cellular Probes. DOI: 10.1016/j.mcp.2014.03.001. Interpretive Summary: Corky root disease of lettuce caused by Sphingobium suberifaciens is an economically important disease in lettuce growing areas worldwide. Infected plants develop yellow to brown lesions on the roots, which can become longitudinal corky ridges that inhibit the flow of nutrients and water to the plant. In severely infested fields, yield losses can reach 30-70% due to the reduction in head size. Researchers have not been able to study the epidemiology of the disease or the disease cycle of the pathogen because the pathogen was difficult to detect and quantify by traditional methods. This research developed sensitive and specific DNA-based detection and quantification methods that detects the pathogen at population levels below those capable of inciting disease and a relationship between disease severity and pathogen population was demonstrated. These methods can now be used to study the pathogen in growers’ fields in order to develop non-chemical disease control methods.
Technical Abstract: The pathogen causing corky root on lettuce, Sphingobium suberifaciens, is recalcitrant to standard epidemiological methods. Primers were selected from 16S rDNA sequences useful for the specific detection and quantification of S. suberifaciens. Conventional (PCR) and quantitative (qPCR) PCR protocols specifically amplified DNA from the type strain of S. suberifaciens (LMG 17323) and related strains but not from other members of the Sphingomonadaceae. As little as 7x104 CFU were detected by PCR, whereas the qPCR protocol detected as little as 2x102 CFU. DNA samples from natural infested soils and diseased lettuce roots were amplified and sequenced fragments were identical or nearly identical to those from S. suberifaciens. In growth chamber experiments, there was a positive correlation between disease severity and S. suberifaciens LMG 17323 populations in roots, as detected by qPCR and detection levels were below population levels of the pathogen from which disease developed.