3.3.65
DESIGN AND UTILIZATION OF A POTATO SOFT ROT ERWINIA-SPECIFIC PCR- BASED DETECTION SYSTEM
PRJ BIRCHl, LJ HYMANl, JR WOOD2 and IK TOTH 1 lFungal and Bacterial Plant Pathology, Scottish Crop Research Institute, Dundee, DD2 5DA, UK. 2Scottish Agricultural Science Agency, East Craigs, Edinburgh, EH12 8NJ, UK. Background and objectives The soft rot erwinias are responsible for disease in a number of crops world wide. The most commercially important of these crops is potato which can be infected by a number of Erwinia carotovora subspecies and Erwinia chrysanthemi pathovars. The elimination of contaminated microplants and minitubers during the early stages of seed potato production is critical to reducing contamination in the later crop. While selective plating on crystal violet pectate (CVP) medium is able to detect most strains, the poor growth of some strains, and/or the overgrowth of saprophytes, can lead to misdiagnosis. A rapid and reliable method to detect all species, subspecies, and strains of the soft rot erwinias responsible for potato disease is thus required for rapid and accurate identification of contaminated stocks. This study describes the design and utilization of PCR primers which detect all E. carotovora subspecies and E. chrysanthemi pathovars in a single PCR reaction while excluding closely related bacterial species. Results and conclusions Sequences from the 16S genes of a number of Erwinia and other Enterobacterial species [1] were aligned and compared. Based on these aligmnents, PCR primers were designed to allow specific amplification of genomic DNA from soft rot erwinias involved in potato disease, while excluding the majority of other Erwinia species and closely related Enterobacterial species. Genomic DNA from all five subspecies of E. carotovora and all seven pathovars of E. chrysanthemi was amplified using the above primers. In addition, DNA from three necrotic Erwinia species were amplified, E. rubrifaciens, E. salicis and E. quercina, although the likelihood of false positives due to the presence of these species is small as none of them are potato pathogens. From a total of over 50 closely related Enterobacterial species including, Escherichia coli, Proteus spp. Serratia spp., Enterobacter spp., and plant and soil saprophytes, no amplification occurred. Detection limits for three soft rot erwinias, Eca, Ecc and Ech pv. 7, in microplants were below 0.003 cells per microplant when a single step PCR reaction was performed. Work is currently ongoing to reduce detection limits by performing two step PCR reactions. In addition to microplants, this PCR-based system is currently being tested on minitubers. References 1. Kwon S-W, Go S-J, Kang W-H, Ryu J-C, Jo J-K. 1997. International Journal of Systematic Bacteriology 47, 1061-1067.