3.3.45
CLASSIFICATION OF PLANT PATHOGENIC PSEUDOMONADS BY GENETIC FINGERPRINTS DERIVED BY THE REPETITIVE EXTRAGENIC PALINDROMIC SEQUENCE POLYMERASE CHAIN REACTION (REP-PCR)

DE STEAD and J HENNESSY

Central Science Laboratory, Sand Hutton, York, Y04 1 LZ, UK

Background and objectives
Several plant pathogenic Pseudomonas species are known to be taxonomically heterogeneous. P. syringae and P. savastanoi comprise approximately 50 pathovars. Differentiation of many of these still relies on host tests which are expensive, time consuming and difficult. Alternative methods for rapid reliable identification are necessary. Our aim was to determine whether one of the repetitive sequence PCR derived fingerprinting methods could be used to differentiate pathovars and provide a more realistic classification of this group of bacteria.

Materials and methods
Approximately 750 strains representing almost all validly described plant pathogenic taxa in Pseudomonas were obtained mainly from the National Collection of Plant Pathogenic Bacteria. All were cultured on nutrient agar for 24 hour's at 28C. REP-PCR fingerprints were prepared [1]. Gels were scanned and the images normalised using Gelcompar software (Applied Mathematics, Kortricht, Belgium). Dendrograms were prepared based on unweighted pair group matching.

Results and conclusions
Most strains gave reproducible fingerprints although for some taxa, some bands (PCR-products) were not present in all strains or repeated profiles. Density of some bands also varied between strains and between repeat runs of strains.

More than 100 clusters of strains were found. In addition many single strains gave unique fingerprints. Some species including P. ficuserectae, P. avellanae, P. amygdaii, P. corrugata, P. fuscovaginae and P. agarici were homogeneous. Others including P. syringae, P. savastanoi, P. cichorii, P. viridiflava, P. marginalis, P. caricapapayae, P. tolaasii, and P. fluorescens were heterogeneous. Within P. syringae, P. savastanoi and to a lesser extent, P. viridiflava and P. cichorii, clusters often correlated with host. Pathovar syringae was particularly heterogeneous although most lilac strains fell into one exclusive cluster. Several other pathovars could not be differentiated from each other. These included pathovars maculicola, tomato, apii and strains from magnoliae and rallistemon. Also, most strains of pathovars coronafaciens, atrofaciens, oryzae and agarcae, mostly from monocots, also clustered together and were not well differentiated from one another.

Pathovars which were well differentiated included delphinii, antirrhini, philadelphi, papulans, aptata, morsprunorum and lachrymans. Pathovar pisi comprised two discrete groups possibly correlated with race classification.

In conclusion, REP-PCR derived genetic fingerprints have great potential value in the classification of this taxonomically difficult group. A revised pathovar-based classification may be appropriate, although at present we cannot use the fingerprints to assign strains to the genomic species proposed by Gardan et al [2].

References
1. Louws FJ, Fulbright DW, Stephens CT and De Bruijn FJ, 1994. Applied and Environmental Microbiology 60, 2286-2295.
2. Gardan L, Shafik H and Grimont PAD, 1997. In P. syringae pathovars and related pathogens (eds. Rudolph K et al) pp 445-448. Kluwyer Academic Publishers, Dordrecht.