Pcr and restriction fragment length polymorphism of a pel gene as a tool to identify erwinia carotovora in relation to potato diseases

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1994, p. 1437-1443

Vol. 60, No. 5

0099-2240/94/$04.00+0 Copyright ©) 1994, American Society for Microbiology

PCR and Restriction Fragment Length Polymorphism of a pel Gene as a Tool To Identify Erwinia carotovora in Relation to Potato Diseases
ARMELLE DARRASSE,' SYLVIE PRIOU,2 ALAIN KOTOUJANSKY,' AND YVES BERTHEAU'* Pathologie Vegetale,Institut National de la Recherche Agronomique INA P-G, 75231 Paris Cedex 05,1 and Pathologie Vegetale, Institut National de la Recherche Agronomique, Domaine de la Motte, 35650 Le Rheu, 2 France
Received 4 October 1993/Accepted 21 February 1994

Using a sequenced pectate lyase-encoding gene (pel gene), we developed a PCR test for Erwinia carotovora. A set of primers allowed the amplification of a434-bp fragment in E. carotovora strains. Among the 89 E. carotovora strains tested, only the Erwinia carotovora subsp. betavasculorum strains were not detected. A restriction fragment length polymorphism (RFLP) study was undertaken on the amplified fragment with seven endonucleases. The Sau3AI digestion pattern specifically identified the Erwinia carotovora subsp. atroseptica strains, and the wholeset of data identified the Erwinia carotovora subsp. wasabiae strains. However, Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. odorifera could not be separated. Phenetic and phylogenic analyses of RFLP results showed E. carotovora subsp. atroseptica as a homogeneous group while E. carotovora subsp. carotovora and E. carotovora subsp. odorifera strains exhibited a geneticdiversity that may result from a nonmonophyletic origin. The use of RFLP on amplified fragments in epidemiology and for diagnosis is discussed.

Pectolytic erwinias cause diseases on a wide range of plants. The species Erwinia carotovora is divided into the four subspecies atroseptica, carotovora, betavasculorum, and wasabiae (13, 20). Erwinia carotovora subsp. betavasculorum and Erwinia carotovorasubsp. wasabiae cause soft rot of sugar beet and Japanese horseradish, respectively (13, 37). Erwinia carotovora subsp. atroseptica is usually restricted to potato under cool temperate climate (27), while Erwinia carotovora subsp. carotovora seems to be more ubiquitous. Some strains, not isolated from potato, have been described as atypical E. carotovora subsp. atroseptica because they exhibit, forexample, the ability to grow at 37°C (24, 29). A new subspecies called Erwinia carotovora subsp. odorifera was proposed for the atypical strains isolated from several hosts, including chicory, that produce odorous volatile metabolites (12). Because of their economic importance, we oriented our study on potato diseases. Symptoms on potato include blanking, blackleg, wilting, and soft rotting ofaerial stem, tuber, and stolon end (28). E. carotovora subsp. atroseptica and E. carotovora subsp. carotovora cause these symptoms under certain field conditions and are found as mixed populations with differential proportions depending on environmental conditions (28, 34). E. carotovora subsp. atroseptica is considered the typical blackleg causal agent in Europe, particularly at the beginning of thegrowing season. Conversely, in pathogenicity tests on potato plants, most E. carotovora subsp. carotovora strains do not produce a typical blackleg symptom at low temperatures (29). However, E. carotovora subsp. carotovora is associated with blackleg of potato in Arizona and Colorado (35). Therefore, it could be interesting to develop identification tests useful in diagnosis. Classical techniquesof bacterial isolation and characterization are time-consuming and relatively insensitive because of
* Corresponding author. Mailing address: Pathologie Wgetale, INRA INA P-G, 16 rue Claude Bernard, 75231 Paris Cedex 05, France. Phone: (33.1) 44 08 17 04. Fax: (33.1) 44 08 17 00. Electronic mail address: bertheau@inapv.inapg.inra.fr.

the high level of saprophytes in the samples (21)....