Characterization of Lactobacillus plantarum from wine must by PCR species-specific and RAPD-PCR G. Spano 1 , L. Beneduce 1 , D. Tarantino 1 , G. Zapparoli 2 and S. Massa 1 1 Istituto di Produzioni e Preparazioni Alimentari, Facolta`di Agraria, Universita`degli Studi di Foggia, Foggia and 2 Dipartimento Scientifico e Tecnologico, Facolta` di Scienze MM.FF.NN., Universita` degli Studi di Verona, Verona, Italy 2002 ⁄ 16: received 8 January 2002, revised 21 May 2002 and accepted 26 June 2002 G. SPANO, L. BENEDUCE, D. TARANTINO, G. ZAPPAROLI AND S. MASSA. 2002. Aims: Physiological and molecular analysis such as PCR species-specific and randomly amplified polymorphic PCR (RAPD-PCR) have been used for typing of Lactobacillus plantarum strains from typical wine must. Methods and Results: Phenotypic tests such as API 50CH and evaluation of D-L-lactate production from glucose were used to perform a preliminary characterization of lactobacilli. Furthermore, 18 strains of lactobacilli were analyzed by PCR species-specific oligonucleotides based on short sequences of the recA gene. Conclusions: Four strains were identified as belonging to the L. plantarum species and were further analysed by RAPD-PCR. The RAPD-PCR profiles were similar in all strains that had positive results for species-specific PCR, suggesting that the four L. plantarum strains were closely related. Significance and Impact of the study: Using PCR species-specific as a preliminary screening test and then RAPD-PCR can be as considered the most reliable method of performing a rapid and correct typing of L. plantarum from wine must. INTRODUCTION Lactobacillus plantarum is a bacterial species that is fre- quently involved in several processes such as plant, meat and fish fermentations and it can often be found as an agent of malolactic fermentation and spoilage activity in wine musts (Lonvaud-Funel 1999). As a malolactic bacterium L. plantarum is responsible for the decrease of wine acidity and improvement of wine taste and flavour. As a spoilage agent L. plantarum can cause increasing volatile acidity and, in some cases, the degradation of tartaric acid leading to a deprecation of the wine. Physiological tests such as the production of CO 2 from glucose, and D-L isomers of lactic acid from glucose, are useful to verify the omo-etero fermentative behaviour of lactic acid bacteria LAB. Furthermore, the definition of a biochemical fermentation profile using API 50 CHL can raise a complete phenotypic characterization of lactobacilli. However, the characterization of L. plantarum is difficult because it is genotypically closely related and phenotypically highly similar to L. pentosus and L. paraplantarum (Curk et al. 1996). Therefore, its correct identification is compli- cated by the ambiguous response of traditional physiological tests. In recent years the availability of molecular tools for studying wine lactobacilli has improved tremendously, allowing better discrimination within the lactobacilli species (Johansson et al. 1995; Zapparoli et al. 2000). Randomly amplified polymorphic DNA (RAPD-DNA) was found to be a useful method to type strains of L. plantarum (Johansson et al. 1995). It is a PCR-based method that uses a single primer under low stringency conditions to amplify fragments of the test organisms, and the fragments amplified may be species or strain specific. Recently, it has been demonstrated that short recA gene sequences can be useful to perform a clear distinction among L. plantarum, L. pentosus and L. paraplantarum (Torriani et al. 2001). The authors designed a set of species-specific PCR primers based on the variable nucleotide sequences of the recA gene. DNA amplification of L. plantarum gave a single band (318 bp), different from those of L. pentosus (218 bp) and L. paraplantarum (107 bp). Correspondence to: Dr G. Spano, Istituto di Produzioni e Preparazioni Alimentari, Facolta`di Agraria, Universita`degli Studi di Foggia, via Napoli, 25, 71100 Foggia, Italy (e-mail: lab.biomol@tiscali.it). ª 2002 The Society for Applied Microbiology Letters in Applied Microbiology 2002, 35, 370–374