Submitted to: Proceedings of the National Academy of Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/15/2006
Publication Date: 10/1/2007
Citation: Li, W., Qijian, S., Brlansky, R., Hartung, J.S. 2007. Genetic diversity of citrus bacterial canker pathogens preserved in herbarium specimens. Proceedings of the National Academy of Sciences. 104(47):18427-18432. Interpretive Summary: Citrus canker disease is one of the most destructive diseases of citrus. Because of the importance of the disease, extensive collections of diseased plant materials were preserved by scientists over the past century as the disease moved out of southern Asia and became established worldwide. The USDA ARS maintains one such large collection in Beltsville MD. These specimens constitute a historical record of the expansion of citrus canker disease and are preserved with records of the date and place of their collection as well as the host on which they were collected. However the bacterial pathogen is dead in such materials and so can not be studied by ordinary microbiological means. We have developed methods to isolate the DNA from these specimens and uniquely identify and distinguish the strains of the pathogen present in the samples. We prepared DNA samples from ninety preserved plant specimens collected from 1914 through 1985 from citrus grown in 34 countries or oceanic atolls. Our results confirm the 90 year old hypothesis that the original introduction of citrus canker to Florida in 1911 was from Japan. We also find several genetically distinct strains of the pathogen in samples from Florida at that period, consistent with multiple independent introductions of the pathogen at that time. Separate analyses of contemporary strains from Florida also indicate multiple independent introductions of the pathogen. Herbaria preserve rich collections of 'fossil' pathogens in the plant tissue. Our methods demonstrate that this material can be used to document the history of plant diseases. Scientists interested in the dissemination and variability with plant pathogens will find our methods useful.
Technical Abstract: Citrus bacterial canker (CBC) caused by Xanthomonas axonopodis pv. citri (Xac) was first documented in India and Java in the mid 19th century. Since that time the known distribution of the disease has steadily increased. Concurrent with the dispersion of the pathogen, the diversity of described strains continues to increase, with novel strains appearing in Saudi Arabia, Iran and Florida in the last decade. Herbarium specimens of infected plants provide an historical record documenting both the geographic distribution and genetic diversity of the pathogen in the past. However no method was available to assess the genetic diversity within these herbarium samples. We have developed a novel method, Insertion Event Scanning (IES), and applied the method to characterize the diversity present within CBC populations documented as herbarium specimens over the past century. IES is based on the specific amplification of junction fragments that define insertion events. The potential for IES in current forensic applications is demonstrated by finding an exact match of pathogen genotypes preserved in herbarium specimens from Japan and Florida, demonstrating the source of the original outbreak of citrus canker in Florida in 1911. IES is a very sensitive technique for differentiating bacterial strains and can be applied to any of the several hundred bacteria for which full genomic sequence data is available.