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Applied and Environmental Microbiology, December 2003, p. 7161-7172, Vol. 69, No. 12
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.12.7161-7172.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Biochemical, Genetic, and Zoosporicidal Properties of Cyclic Lipopeptide Surfactants Produced by Pseudomonas fluorescens

Jorge T. de Souza,1 Marjan de Boer,2 Pieter de Waard,3 Teris A. van Beek,4 and Jos M. Raaijmakers1*

Laboratory of Phytopathology, Department of Plant Sciences, Wageningen University, 6709 PD Wageningen,1 Section Flower Bulbs, Applied Plant Research, 2160 AB Lisse,2 Wageningen NMRCentre, Wageningen University, 6700 ET Wageningen,3 Natural Products Chemistry Group, Laboratory of Organic Chemistry, Wageningen University, 6703 HB Wageningen, The Netherlands4

Received 5 June 2003/ Accepted 4 September 2003

Zoospores play an important role in the infection of plant and animal hosts by oomycetes and other zoosporic fungi. In this study, six fluorescent Pseudomonas isolates with zoosporicidal activities were obtained from the wheat rhizosphere. Zoospores of multiple oomycetes, including Pythium species, Albugo candida, and Phytophthora infestans, were rendered immotile within 30 s of exposure to cell suspensions or cell culture supernatants of the six isolates, and subsequent lysis occurred within 60 s. The representative strain SS101, identified as Pseudomonas fluorescens biovar II, reduced the surface tension of water from 73 to 30 mN m-1. The application of cell suspensions of strain SS101 to soil or hyacinth bulbs provided significant protection against root rot caused by Pythium intermedium. Five Tn5 mutants of strain SS101lacked the abilities to reduce the surface tension of water and to cause lysis of zoospores. Genetic characterization of two surfactant-deficient mutants showed that the transposons had integrated into condensation domains of peptide synthetases. A partially purified extract from strain SS101 reduced the surface tension of water to 30 mN m-1 and reached the critical micelle concentration at 25 µg ml-1. Reverse-phase high-performance liquid chromatography yielded eight different fractions, five of which had surface activity and caused lysis of zoospores. Mass spectrometry and nuclear magnetic resonance analyses allowed the identification of the main constituent as a cyclic lipopeptide (1,139 Da) containing nine amino acids and a 10-carbon hydroxy fatty acid. The other four zoosporicidal fractions were closely related to the main constituent, with molecular massesranging from 1,111 to 1,169 Da.

* Corresponding author. Mailing address: Laboratory of Phytopathology, Department of Plant Sciences, Wageningen University (WU), Binnenhaven 5, P.O. Box 8025, 6709 PD Wageningen, The Netherlands. Phone: 31 317 483427. Fax: 31 317 483412. E-mail: jos.raaijmakers{at}wur.nl.

Applied and Environmental Microbiology, December 2003, p. 7161-7172, Vol. 69, No. 12
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.12.7161-7172.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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