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Seventy bacterial isolates from the rhizosphere of tomato were screened for antagonistic activity against the tomato foot and root rot-causing fungal pathogen Fusarium oxysporum f. sp. radicis-lycopersici. One isolate, strain PCL1391, appeared to be an efficient colonizer of tomato roots and an excellent biocontrol strain in an F. oxysporum/tomato test system. Strain PCL1391 was identified as Pseudomonas chlororaphis and further characterization showed that it produces a broad spectrum of antifungal factors (AFFs), including a hydrophobic compound, hydrogen cyanide, chitinase(s), and protease(s). Through mass spectrometry and nuclear magnetic resonance, the hydrophobic compound was identified as phenazine-1-carboxamide (PCN). We have studied the production and action of this AFF both in vitro and in vivo. Using a PCL1391 transposon mutant, with a lux reporter gene inserted in the phenazine biosynthetic operon (phz), we showed that this phenazine biosynthetic mutant was substantially decreased in both in vitro antifungal activity and biocontrol activity. Moreover, with the same mutant it was shown that the phz biosynthetic operon is expressed in the tomato rhizosphere. Comparison of the biocontrol activity of the PCN-producing strain PCL1391 with those of phenazine-1-carboxylic acid (PCA)-producing strains P. fluorescens 2-79 and P. aureofaciens 30-84 showed that the PCN-producing strain is able to suppress disease in the tomato/F. oxysporum system, whereas the PCA-producing strains are not. Comparison of in vitro antifungal activity of PCN and PCA showed that the antifungal activity of PCN was at least 10 times higher at neutral pH, suggesting that this may contribute to the superior biocontrol performance of strain PCL1391 in the tomato/F. oxysporum system.

To study colonization of the tomato root system, we previously have described a gnotobiotic quartz sand system, in which seedlings inoculated with one or two bacterial strains were allowed to grow. Here we present a scanning electron microscope description of the colonization of the tomato root system by Pseudomonas fluorescens biocontrol strain WCS365, with emphasis on spatial-temporal colonization patterns, based on an improved scanning electron microscopy procedure. Upon inoculation of the germinated seed, proliferation on the seed coat was observed for 2 to 3 days. Within 1 to 3 days, micro-colonies developed, mainly at the root base. Most micro-colonies were localized in junctions between epidermal root cells, whereas others were found in indented parts of the epidermal surface. Downward to the root tip, only single bacterial cells were found. Colonization progressed down the root, initially as single cells. A semi-transparent film appeared to enclose the root surface and micro-colonies present on the root. After 7 days, micro-colonies had developed at positions where only single cells were observed previously and distribution of the bacteria along the root varied from ≈106 CFU per cm of root near the root base to ≈102 to 103 CFU per cm of root near the root tip. Similar colonization patterns were found for the P. fluorescens biocontrol strains CHA0 and F113, and P. putida strain WCS358, as well as for four species that have repeatedly been isolated from tomato roots from a commercial tomato field near Granada, Spain. In contrast, four Rhizobium strains and one Acinetobacter radioresistens strain showed poor colonization and micro-colonies were not observed. Based on the described data, we present a model for colonization of the deeper root parts after seed inoculation by P. fluorescens biocontrol strains, in which single cells occasionally establish on a deeper root section where they sometimes develop into micro-colonies. We hypothesize that micro-colonies are the sites where the intracellular N-acyl-L-homoserine lactone concentration is sufficiently high to cause maximal production of biocontrol factors such as antibiotics and exoenzymes and that micro-colonies explain the relatively high conjugation frequency observed between pseudomonads in the rhizosphere.

Colonization-defective, transposon-induced mutants of the efficient root colonizer Pseudomonas fluorescens WCS365 were identified with a gnotobiotic system. Most mutants were impaired in known colonization traits, i.e., prototrophy for amino acids, motility, and synthesis of the O-antigen of LPS (lipopolysaccharide). Mutants lacking the O-antigen of LPS were impaired in both colonization and competitive growth whereas one mutant (PCL1205) with a shorter O-antigen chain was defective only in colonization ability, suggesting a role for the intact O-antigen of LPS in colonization. Eight competitive colonization mutants that were not defective in the above-mentioned traits colonized the tomato root tip well when inoculated alone, but were defective in competitive root colonization of tomato, radish, and wheat, indicating they contained mutations affecting host range. One of these eight mutants (PCL1201) was further characterized and contains a mutation in a gene that shows homology to the Escherichia coli nuo4 gene, which encodes a subunit of one of two known NADH:ubiquinone oxidoreductases. Competition experiments in an oxygen-poor medium between mutant PCL1201 and its parental strain showed a decreased growth rate of mutant PCL1201. The requirement of the nuo4 gene homolog for optimal growth under conditions of oxygen limitation suggests that the root-tip environment is micro-aerobic. A mutant characterized by a slow growth rate (PCL1216) was analyzed further and contained a mutation in a gene with similarity to the E. coli HtrB protein, a lauroyl transferase that functions in lipid A biosynthesis.

Efficient colonization of the plant root is thought to be crucial for the plant‐beneficial effect of particular Pseudomonas strains. Since root colonization is often the limiting step for successful plant growth stimulation, this process needs improvement. It is therefore important to acquire more information as regards (i) the conditions in the rhizosphere, and (ii) the bacterial traits that are involved in colonization. This review discusses some recent studies that focus on these two issues.