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

Microbial compounds that alter the conditions prevailing at a surface or interface are often referred to as adjuvants, bioemulsifiers, or biosurfactants (5, 14, 51). A variety of microorganisms, including bacteria, fungi, and yeasts, have been reported to produce biosurfactants (16, 36). Several of these biosurfactants are well described chemically and categorized into high- and low-molecular-mass compounds. The low-molecular-mass biosurfactants include glycolipids and lipopeptides, such as rhamnolipids and surfactin. The high-molecular-mass compounds include proteins and lipoproteins, or complex mixtures of these polymers. Although biosurfactants are structurally diverse, they all have an amphiphilic nature, i.e., they contain both hydrophobic and hydrophilic groups. The hydrophobic moieties are usually saturated, unsaturated, or hydroxylated fatty acids or apolar amino acids, like leucin and isoleucin. The hydrophilic moieties consist of mono-, di-, or polysaccharides, carboxylic acids, polar amino acids, or peptides (36).
Among the bacterial genera, Pseudomonas spp. have been reported to produce biosurfactants (14, 33, 36). Pseudomonas spp. are common inhabitants of soil and rhizosphere environments and have received considerable attention in the areas of bioremediation of xenobiotics and biological control of plant-pathogenic fungi. In the area of bioremediation, surfactant-producing Pseudomonas spp. have been implicated in facilitating the degradation of ubiquitous pollutants, such as polycyclic aromatic hydrocarbons and n-alkanes (2, 12). In the area of biological control of plant-pathogenic fungi, the potential of biosurfactants produced by Pseudomonas spp. was recently recognized. Rhamnolipids produced by strains of Pseudomonas aeruginosa were shown to be highly effective against plant pathogens, including Pythium aphanidermatum, Plasmopara lactucae-radicis, and Phytophthora capsici (43). Purified rhamnolipids caused cessation of motility and the lysis of entire zoospore populations within <1 min. The introduction of a rhamnolipid-producing strain into a recirculating hydroponic system gave good, although transient, control of P. capsici on pepper (43). Kim et al. (27) confirmed and extended these observations by showing that rhamnolipid B, produced by P. aeruginosa B5, has not only lytic effects on zoospores but also inhibitory activity against the spore germination and hyphal growth of several other pathogens. Mycelial growth of P. capsici and spore germination of Colletotrichum orbiculare were inhibited in vitro, and the diseases caused by these pathogens were suppressed in pepper and cucumber plants, respectively, by application of purified rhamnolipid B to leaves (27).
Several cyclic lipopeptide surfactants with antibiotic properties were recently proposed as biological compounds for the control of plant-pathogenic fungi (29, 31-33, 48). Viscosinamide, produced by soil-inhabiting Pseudomonas sp. strain DR54, was shown to induce encystment of Pythium zoospores and to adversely affect mycelia of Rhizoctonia solani and Pythium ultimum, causing reduced growth and intracellular activity, hyphal swellings, increased branching, and rosette formation (18, 46, 47). The specific cyclic lipopeptide amphisin, produced by Pseudomonas sp. strain DSS73, appeared to play an important role in the surface motility of the producing strain, allowing efficient containment of root-infecting plant-pathogenic fungi (1). Furthermore, in combination with cell wall-degrading enzymes of Trichoderma atroviride, lipodepsipeptides produced by the pathogen Pseudomonas syringae pv. syringae acted synergistically in antagonism to various plant-pathogenic fungi (15). Collectively, these studies clearly indicate the potential of biosurfactant-producing bacteria for crop protection.
In this study, surfactant-producing Pseudomonas isolates were obtained from the rhizosphere of wheat and screened for activity against zoospores of multiple oomycete pathogens, including Pythium species and Phytophthora infestans. The biocontrol ability of the representative strain Pseudomonas fluorescens SS101 was tested in hyacinth flower bulbs against root rot caused by Pythium intermedium. Two genes involved in surfactant production by P. fluorescens strain SS101 were obtained by random Tn5 mutagenesis followed by anchored PCR and subsequent sequencing of the Tn5 flanking regions. Tn5 mutants were characterized phenotypically, and their activities against zoospores of oomycete pathogens were compared to that of their parental strain. The surfactants produced by strain SS101 were isolated by reverse-phase high-pressure liquid chromatography (RP-HPLC), and their activities were assessed in bioassays. The identity of the main surface-active constituent was determined by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR).
UU, P. A. H. M. Bakker, Section Plant Pathology, Utrecht University (Utrecht, The Netherlands); DSMZ, Deutsche Sammlung von Mikroorganismen und Zelculturen (Braunschweig, Germany); PPO, Applied Plant Research, Section Flower Bulbs (Lisse, The Netherlands); CBS, Dutch Collection of Microorganisms (Utrecht, The Netherlands); WU, Laboratory of Phytopathology, Wageningen University (Wageningen, The Netherlands).
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