Letters in Applied Microbiology 1997, 24, 249–252 Detection of Pseudomonas putida B in the rhizosphere by RAPD B.M. Hansen and A. Winding Department of Marine Ecology and Microbiology, National Environmental Research Institute, Roskilde, Denmark 1229/96: received 17 July 1996 and accepted 14 August 1996 B.M. HANSEN AND A. WINDING. 1997. A method was developed for the detection of Pseudomonas putida B MM12 released into the rhizosphere of non-sterile barley, using a Random Amplified Polymorphic DNA (RAPD)-generated probe for hybridization with RAPD products generated from DNA extracted from the rhizosphere. The detection procedure involves extraction of rhizosphere bacteria by sonication, extraction of DNA by boiling, RAPD and Southern hybridization with RAPD products and the selected probe. The level of detection of MM12 was at least 1·9 × 10 4 cells g −1 barley root. MM12 was detected in rhizosphere when it constituted as little as 0·5% of the culturable population. INTRODUCTION MATERIALS AND METHODS Identification of specific micro-organisms in environmental Bacterial strains samples is important for risk analysis of released bacteria. Molecular biological techniques provide highly specific Ten Pseudomonas strains were used : five Ps. putida B, MM1, MM3, MM11, MM12 and MM12 R ; three Ps. fluorescens I, methods for the detection and identification of such bacteria. The application of Random Amplified Polymorphic DNA MM2, MM7 and DSM50090 ; and two Ps. fluorescens II, MM6 and Agl. The MM strains were isolated from barley (RAPD) (Welsh and McClelland 1990 ; Williams et al. 1990) for production of isolate-specific DNA fingerprints is roots and Agl from soil. MM12 R was a spontaneous rifam- picin-resistant mutant of MM12, isolated in this study. Bac- especially promising (Hadrys et al. 1992). This technique has the advantages that no DNA sequence information of the teria were cultivated overnight at room temperature in Luria Bertani medium (LB ; Sambrook et al. 1989). When selecting organism is needed, and bacteria do not have to be genetically marked. In addition, polymorphic RAPD fragments have for MM12 R , the medium was supplemented with rifampicin (100 mg ml −1 in agar plates and 50 mg ml −1 in liquid medium). been used for the development of isolate- and species-specific probes for bacteria (Fani et al. 1993 ; Manulis et al. 1994), fungi (Dobrowolski and O’Brien 1993 ; Potenza et al. 1994), protozoa (Bartholomew et al. 1995) and plants (Aitken et al. Growth and harvest of barley roots 1994) for distinguishing between closely related organisms. A sandy loam from Roskilde, Denmark, was sieved (5·6 mm However, the reported applications of the RAPD probes mesh size) and stored at 4°C for 1 month before use. The were primarily for the identification of cultured organisms, soil was distributed into plastic pots (diameter 12 cm) with while the application of RAPD-based probes for the detection ca 750 g in each. Undressed seeds of winter barley were of organisms directly from a natural environment is rare. The inoculated with an overnight culture of 8×10 9 cfu ml −1 aim of the present study was to design a specific probe for MM12 R , which was washed and resuspended in Winogradsky Pseudomonas putida B MM12 by the RAPD technique and to (W) salt solution (Holm and Jensen 1972). The seeds were test the specificity and ability of the probe to detect the left in the bacterial suspension (control seeds in W) for 20 bacterium in the rhizosphere of barley. min at room temperature, shaken to remove liquid, and 12 seeds were transferred to each pot. The pots were placed in an incubator with a 12 h light (20°C) and 12 h dark (15°C) Correspondence to: Dr B. M. Hansen, Department of Marine Ecology and cycle, and were watered from underneath with tap water. Microbiology, National Environmental Research Institute, PO Box 358, Frederiksborgvej 399, DK-4000 Roskilde, Denmark (e-mail: bmh@dmu.dk) After 2 weeks, the plants were harvested and the soil carefully © 1997 The Society for Applied Bacteriology