Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/20/2005
Publication Date: 8/15/2005
Citation: Li, W., Brlansky, R., Hartung, J.S. 2005. Amplification of dna of xanthomonas axonopodis pv citri from historic citrus canker herbarium specimens. Journal of Microbiological Methods. 65:237-246.
Interpretive Summary: Museums and botanical herbaria around the world maintain millions of specimens of plants collected over hundreds of years. Some of these specimens show clear symptoms of plant diseases that cause significant problems today. These specimens could provide valuable clues about the origins and early distribution of such diseases if they were studied using powerful DNA-based methods. Unfortunately, these specimens contain powerful inhibitors of the enzymes needed for DNA analysis and the DNA itself is broken into many small pieces. We have developed methods to purify such ‘historic DNA’ and study it using DNA-based methods. For our work we have used a large collection of citrus specimens nearly a century old that show clear symptoms of the bacterial disease citrus canker. We have developed methods for extracting DNA from these specimens with excellent purity. We have also used the full-genome sequence of the citrus canker pathogen to design assays that specifically identify the pathogen in historic plant samples. By combining a large panel of extraction methods with a large panel of assays, we have identified the best methods for both the extraction and analysis of DNA from these valuable specimens. Our methods will be used to trace the origins and evolution of the citrus canker pathogen over the past century. These studies may also provide insight on the continual emergence of new forms of this very important plant disease, which threatens the U.S. citrus industry and will be useful to both the scientific and plant disease regulatory communities.
Technical Abstract: Herbaria are important resources for the study of the origins and dispersal of plant pathogens, particularly bacterial plant pathogens that incite local lesions in which large numbers of pathogen genomes are concentrated. Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus bacterial canker disease, is a notable example of such a pathogen. The appearance of novel strains of the pathogen in Florida and elsewhere make it increasingly important to understand the relationships among strains of this pathogen. USDA-ARS at Beltsville, Maryland maintains approximately 700 herbarium specimens with citrus canker disease lesions up to 90 years old, originally collected from all over the world, and so is an important resource for phytogeographic studies of this bacterium. Unfortunately, DNA in herbarium specimens is degraded and may contain high levels of inhibitors of PCR. In this study, we compared a total of 23 DNA isolation techniques in combination with 31 novel primer pairs in order to develop an efficient protocol for the analysis of Xac DNA in herbarium specimens. We identified the most reliable extraction method, identified in terms of successful amplification by our panel of 31 primer pairs. We also identified the most robust primer pairs, identified as successful in the largest number of extracts prepared by different methods. We amplified Xac genomic sequences up to 542 bp long from herbarium samples up to 89 years old. Primers varied in effectiveness, with some primer pairs amplifying Xac DNA from a 1/10,000 dilution of extract from a single lesion from a citrus canker herbarium specimen. Our methods will be useful to identify pathogens and perform molecular analyses of bacterial and possibly fungal organisms from herbarium specimens.