3.3.43
PCR DETECTION OF ERWINIA CAROTOVORA SSP. CAROTOVORA FROM VARIOUS PLANTS IN POLAND

L Z0TOBOWSKA and H POSPIESZNY

lnstitute of Plant Protection, Mtczunna 20, 60-318 Poznan, Poland

Background and abjectives
Soft rot of vegetables and potato may be caused by various bacterial plant pathogens. Economically most important species belong to the genus Erwinia and less extensively studied group of pectolytic Pseudomonas. Genus Erwinia formally is divided into species and subspecies with Erwinia carotovora ssp.atroseptica (Eca), Erwinia carotovora ssp. carotovora (Ecc), and Erwinia chrysanthemi (Ech) as the most important pathogens, but in fact soft rot erwinias form more complex group. There are 5 Eca serogroups, about 40 Ecc serogroups and 6 pathovars or 9 biovars of Ech. The diagnostics of this group of bacteria is therefore very complicated. The most commonly used biochemical and aerological methods are very time and labor consuming and the results are often unsatisfactory due to the above mentioned diversity of soft rot erwinias. Currently the PCR seemes to be the most promissing method in diagnostics of bacterial plant pathogens because of its speed, specificity and sensitivity. The heterogeneity of soft rot erwinias however influences specificity of primers. There are several workers who attempted to develope primers sc for Eca, Ecc and Ech, respectively. The attempts are more successful in case of Eca and Ech[31, but primers specific for Ecc were as yet not reported. The objective of our work was to design and check E. carotovora-specific and Ecc-specific primers and use them in identification and differentiation of 250 polish Erwinia ssp.isolates.

Materials and methods
PCR primers used in this study were designed on the basis of published sequences of Erwinia genes. The sequences of Ecc-specific primers are: PECC 5'-AAC TCG M GCC TGA TTG A-3', pECCRev 5'-TGT CAC CGC TAC AGC TCA GA-3'(pchl-pel3 gene sequene [21) and E. carotovora-specific primers are: PECAI 5'-CCG CGA AAG TGG ATT CAA AAG G-3', pECA2 5'-TGC TTG CGC CGC TTA GAC C-3' (pelb gene sequence [11). The thermocycler was programmed as followes: 94'C for 4 min-, 30 cycles at 94'C for 1 min., 60'C for 1 min., 72'C for 2 min. and ly one cycle at 940C for 1 min., 60c'C for 1 min. and 72'C for 10 min.

Results and conclusions
The specificity of both primer sets was thoroughly checked using several bacterial isolates belonging to various taxonomical groups, including pectolytic Pseudomonas spp.. Primers proved to be specific within the genus Erwinia. Ecc-specific primer set was also checked against 15 representatives of different Ecc serogroupes kindly provided by M.C.M. Perombelon and 36 and 4 representatives of Ecc and Eca serogroupes respectively kindly provided by S.H. De Boer. Only 2 of the above mentioned 15 Ecc strains and 5 of 36 Ecc strains were not recognised by our printers, whereas negative results was obtained with 4 Eca strains and also with 2 Ech strains kindly provided by E.Lojkowska. During our study we obtained 250 isolates of soft rot erwinias from potato, carrot. parsley, celery, cabbage, onion and leek. Using biochemical and serological methods, 71 of these isolates were identified as Eca, 150 as Ecc and 29 as biochemically atypical erwinias. All of biochemically typical and atypical E. carotovora isolates were recognised by ECAI-ECA2 primer se@ whereas 2 Ech strains gave negative results with these primers. 133 (891/o) of typical Ecc isolates were recogmzed by ECC-ECCRev primers and also about 50% of isolates belonging to atypical group was recognised by the primers as Ecc. Thus the specificity of ECC-ECCRev primers regarding Ecc proved comparatively high, therefore the use of PCR method in evaluation of health status (contamination level) of storage organs of several econonucally important vegetables becomes possible. During our study we isolated also 76 isolates of pectolytic fluorescent Pseudomonas. Their role in soft rot pathogenesis in Poland and economical importance will be evaluated in the near future.

References
1. Lei S.P., Lim H.C., Wang S.S., Cafloway J.,Wilcox G. 1987. J. Bacteriol. 169:4379-4383.
2.. Liu Y., Chatteijee A-, Chattedee AK. 1994.. Appl. Environ. Mcrobiol. 60: 2545-2552.
3. Sn-iid E.J., Jansen A.H.J., Gorris L.G.M. 1995. Plant Path. 44..1058-1069.