b>IDENTIFICATION OF PECTOLYTIC ERWINIAS ISOLATED FROM INFECTED POTATO PLANTS IN POLAND
b>IDENTIFICATION OF PECTOLYTIC ERWINIAS ISOLATED FROM INFECTED POTATO PLANTS IN POLAND W SLEDZl, S JAFRAl, M WALERONl, IK TOTH2, LJ HYMAN2, MCM PEROMBELON 2 and E LOJKOWSKA 1 lLaboratory of Phytopathology, Department of Biotechnology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland. 2Scottish Crop Research Institute, lnvergowrie, Dundee DD2 5DA, Scotland. Background and objectives The genus Erwinia contains several pectolytic species (Erwinia carotovora including subspecies atroseptica - Eca, carotovora - Ecc, wasabiae - Ecw, betavasculorum - Ecb and ododfera - Eco, and Erwinia chrysanthemi - Echr), which cause disease symptoms in many different plant species. Potato is one of the most important agricultural species attacked by these erwinias. However, the presence of bacteria on or within a plant does not always lead to disease development. Often, a lethal infection is only revealed under favourable conditions for disease development, causing significant problems in selection and elimination of infected material. In order to identify infected plants or tubers, therefore, it is important to determine the presence and number of Erwinia cells in a sample, in addition to any visible disease symptoms which may be present. This can be acheived using specific detection systems. However, since no one system is able to target all soft rot erwinias, e.g. current monoclonal antibody-based systems detect Eca serogroup 1 only, the system(s) applied will depend on the presence and distribution of the different soft rot erwinias in any particular area or sample type. In this study, we determined the types of Erwinia present in Poland, and their distribution, prior to implementing detection systems for large-scale screening of plant material. Results and conclusions About 230 samples of potato plants with blackleg and soft rot symptoms were obtained from Control Inspectors from the State Inspectorate of Plant Protection of Poland. Pectolytic bacteria, typically forming deep cup-like cavities, were isolated on Crystal Violet Pectate medium. For estimation of Erwinia subspecies we used biochemical, serological and molecular methods - polymerase chain reaction (PCR). Almost 700 bactehal isolates were identified. Biochemical tests allowed for preliminary identification of 95% of the examined strains. PCR was then used to confirm the results obtained from biochemical analyses and also to identify other soft rot erwinias not identified by biochemical analysis. In a multiplex PCR, specific primers were used for Eca and primers based on the sequences of genes from the peiY family were used for Ecc. Results were compared with biochemical tests. For determination of Eca serotype 1, an ELISA test with monocional antibodies was used. It was estimated that 58% 67%, and 50% of isolated strains from Northern Poland, Central Poland and Southern Poland, respectively, were Eca. About 61% of isolates from plants with blackleg and 51% from tubers with soft rot symptoms were Eca. It was estimated that only 71% of Eca strains belonged to serotype 1. However, serotype 1 constituted 100% of tuber isolates and 67% of stem isolates. For Ech determination, specific primers were used based on the pei genes . New primers based on the peiL gene , were checked for their specificity to Ech. Almost 100 different Erwinia (Eca, Ecc, Ecw, Ecb, Eco, Ech) strains from Polish, Scottish and French collections, and isolates from infected plants, were analysed with the primers based on the peiL gene. Only those belonging to Erwinia chrysanthemi gave a specific product. No positive results were obtained with these primers for any strains isolated from the Polish area. References 1. Nassar A, Darrasse A, Lemattre M, Kotoujansky A, Dervin C, Vedel R, Bertheau Y, 1996. Applied and Environmental Microbiology 62, 2228-35. 2. Lojkowska E, Masclaux C, Boccara M, Robert-Baudouy J, Hugouvieux-Cotte-Pattat N, 1995. Molecular Microbiology 16, 1183-95.