Guido V. Bloemberg

Molecular basis of plant growth promotion and biocontrol by rhizobacteria

Plant-growth-promoting rhizobacteria (PGPRs) are used as inoculants for biofertilization, phytostimulation and biocontrol. The interactions of PGPRs with their biotic environment, for example with plants and microorganisms, are often complex. Substantial advances in elucidating the genetic basis of the beneficial effects of PGPRs on plants have been made, some from whole-genome sequencing projects. This progress will lead to a more efficient use of these strains and possibly to their improvement by genetic modification.

Rhizoremediation: a beneficial plant-microbe interaction.

Worldwide, contamination of soil and ground water is a severe problem. The negative effects of pollutants on the environment and on human health are diverse and depend on the nature of the pollution. The search for alternative methods for excavation and incineration to clean polluted sites resulted in the application of bioremediation techniques. In this review, we describe some generally accepted bioremediation tools and subsequently focus on the combination of two approaches, phytoremediation and bioaugmentation, resulting in rhizoremediation. During rhizoremediation, exudates derived from the plant can help to stimulate the survival and action of bacteria, which subsequently results in a more efficient degradation of pollutants. The root system of plants can help to spread bacteria through soil and help to penetrate otherwise impermeable soil layers. The inoculation of pollutant-degrading bacteria on plant seed can be an important additive to improve the efficiency of phytoremediation or bioaugmentation.

Flagella-Driven Chemotaxis Towards Exudate Components Is an Important Trait for Tomato Root Colonization by Pseudomonas fluorescens

Motility is a major trait for competitive tomato root-tip colonization by Pseudomonas fluorescens. To test the hypothesis that this role of motility is based on chemotaxis toward exudate components, cheA mutants that were defective in flagella-driven chemotaxis but retained motility were constructed in four P. fluorescens strains. After inoculation of seedlings with a 1:1 mixture of wild-type and nonmotile mutants all mutants had a strongly reduced competitive root colonizing ability after 7 days of plant growth, both in a gnotobiotic sand system as well as in nonsterile potting soil. The differences were significant on all root parts and increased from root base to root tip. Significant differences at the root tip could already be detected after 2 to 3 days. These experiments show that chemotaxis is an important competitive colonization trait. The best competitive root-tip colonizer, strain WCS365, was tested for chemotaxis toward tomato root exudate and its major identified components. A chemotactic response was detected toward root exudate, some organic acids, and some amino acids from this exudate but not toward its sugars. Comparison of the minimal concentrations required for a chemotactic response with concentrations estimated for exudates suggested that malic acid and citric acid are among major chemo-attractants for P. fluorescens WCS365 cells in the tomato rhizosphere.

Biocontrol by Phenazine-1-carboxamide-Producing Pseudomonas chlororaphis PCL1391 of Tomato Root Rot Caused by Fusarium oxysporum f. sp. radicis-lycopersici

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.

Root colonization by phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 is essential for biocontrol of tomato foot and root rot

The phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicislycopersici. To test whether root colonization is required for biocontrol, mutants impaired in the known colonization traits motility, prototrophy for amino acids, or production of the site-specific recombinase, Sss/XerC were tested for their root tip colonization and biocontrol abilities. Upon tomato seedling inoculation, colonization mutants of strain PCL1391 were impaired in root tip colonization in a gnotobiotic sand system and in potting soil. In addition, all mutants were impaired in their ability to control tomato foot and root rot, despite the fact that they produce wild-type levels of phenazine-1-carboxamide, the antifungal metabolite previously shown to be required for biocontrol. These results show, for what we believe to be the first time, that root colonization plays a crucial role in biocontrol, presumably by providing a delivery system for antifungal metabolites. The ability to colonize and produce phenazine-1-carboxamide is essential for control of F. oxysporum f. sp. radicis-lycopersici. Furthermore, there is a notable overlap of traits identified as being important for colonization of the rhizosphere and animal tissues.

Simultaneous Imaging of Pseudomonas fluorescens WCS365 Populations Expressing Three Different Autofluorescent Proteins in the Rhizosphere: New Perspectives for Studying Microbial Communities

To visualize simultaneously different populations of pseudomonads in the rhizosphere at the single cell level in a noninvasive way, a set of four rhizosphere-stable plasmids was constructed expressing three different derivatives of the green fluorescent protein (GFP), namely enhanced cyan (ECFP), enhanced green (EGFP), enhanced yellow (EYFP), and the recently published red fluorescent protein (RFP; DsRed). Upon tomato seedling inoculation with Pseudomonas fluorescens WCS365 populations, each expressing a different autofluorescent protein followed by plant growth for 5 days, the rhizosphere was inspected using confocal laser scanning microscopy. We were able to visualize simultaneously and clearly distinguish from each other up to three different bacterial populations. Microcolonies consisting of mixed populations were frequently observed at the base of the root system, whereas microcolonies further toward the root tip predominantly consisted of a single population, suggesting a dynamic behavior of microcolonies over time. Since the cloning vector pME6010 has a broad host range for gram-negative bacteria, the constructed plasmids can be used for many purposes. In particular, they will be of great value for the analysis of microbial communities, for example in processes such as biocontrol, biofertilization, biostimulation, competition for niches, colonization, and biofilm formation.

Microbe-plant interactions: principles and mechanisms

The present status of research on the molecular basis of microbe–plant interactions is discussed. Principles and mechanisms which play a role in the interactions of microbial pathogens, biofertilizers, phytostimulators, rhizoremediators and biocontrol agents with the plants are treated. Special emphasis is given to colonization, phase variation, two-component systems, quorum sensing, complex regulation of the syntheses of extracellular enzymes and secondary metabolites, Type 4 pili and Type III and Type IV secretion systems.

Characterization of two Pseudomonas putida lipopeptide biosurfactants, putisolvin I and II, which inhibit biofilm formation and break down existing biofilms

Pseudomonas putida strain PCL1445 was isolated from roots of plants, grown on a site polluted with polycyclic aromatic hydrocarbons. PCL1445 produces biosurfactant activity at the end of the exponential growth phase. High‐performance liquid chromatography (HPLC) analysis of supernatant extracts of PCL1445 showed two peaks with surface‐tension reducing activity, tentatively assigned as biosurfactants putisolvin I and putisolvin II and was followed by structural analyses. A transposon mutant of PCL1445, strain PCL1436, which lacks the two surface‐active peaks appeared to be mutated in an open reading frame (ORF) with amino acid homology to various lipopeptide synthetases. Structural analyses of the two biosurfactants of PCL1445 revealed that both are novel cyclic lipodepsipeptides with a hexanoic lipid chain connected to the N‐terminus of a 12‐amino‐acid peptide moiety, in which the C‐terminal carboxylic acid group forms an ester with the hydroxyl side‐chain of Ser9. The difference between the two structures is located in the second amino acid from the C‐terminus, being valine for putisolvin I, and leucine/isoleucine for putisolvin II. We show that these novel compounds lower the surface tension and influence the biofilm development on polyvinyl chloride (PVC). Biofilm formation of the bio‐synthetic mutant PCL1436 was strongly increased containing more cells, which formed aggregates earlier as compared with wild‐type PCL1445 biofilms. Using purified putisolvin I and II it was shown that biofilm formation of different Pseudomonas strains was inhibited and most interestingly, that both putisolvins are also able to break down existing Pseudomonas biofilms.

Novel aspects of tomato root colonization and infection by Fusarium oxysporum f. sp. radicis-lycopersici revealed by confocal laser scanning microscopic analysis using the green fluorescent protein as a marker

The fungus Fusarium oxysporum f. sp. radicis-lycopersici is the causal agent of tomato foot and root rot disease. The green fluorescent protein (GFP) was used to mark this fungus in order to visualize and analyze the colonization and infection processes in vivo. Transformation of F oxysporum f. sp. radicis-lycopersici was very efficient and gfp expression was stable for at least nine subcultures. Microscopic analysis of the transformants revealed homogeneity of the fluorescent signal, which was clearly visible in the hyphae as well as in the chlamydospores and conidia. To our knowledge, this is the first report in which this is shown. The transformation did not affect the pathogenicity. Using confocal laser scanning microscopy, colonization, infection, and disease development on tomato roots were visualized in detail and several new aspects of these processes were observed, such as (i) the complete colonization pattern of the tomato root system; (ii) the very first steps of contact between the fungus and the host, which takes place at the root hair zone by mingling and by the attachment of hyphae to the root hairs; (iii) the preferential colonization sites on the root surface, which are the grooves along the junctions of the epidermal cells; and (iv) the absence of specific infection sites, such as sites of emergence of secondary roots, root tips, or wounded tissue, and the absence of specific infection structures, such as appressoria. The results of this work prove that the use of GFP as a marker for F. oxysporum f. sp. radicis-lycopersici is a convenient, fast, and effective approach for studying plant-fungus interactions.

Selection of a plant-bacterium pair as a novel tool for rhizostimulation of polycyclic aromatic hydrocarbon-degrading bacteria.

We developed a novel procedure for the selection of a microbe-plant pair for the stable and efficient degradation of naphthalene. Based on the rationale that root exudate is the best nutrient source available in soil, the grass (Lolium multiflorum) cultivar Barmultra was selected because of its abilities to produce a highly branched root system, root deeply, and carry a high population of Pseudomonas spp. bacteria on its roots. Starting with a mixture of total rhizobacteria from grass-like vegetation collected from a heavily polluted site and selecting for stable naphthalene degradation as well as for efficient root colonization, Pseudomonas putida strain PCL1444 was isolated. The strains ability to degrade naphthalene was shown to be stable in the rhizosphere. Moreover, it had superior root-colonizing properties because, after the inoculation of grass seedlings, it appeared to colonize the root tip up to 100-fold better than the efficient root colonizer Pseudomonas fluorescens WCS365. Strain PCL1444 uses root exudate as the dominant nutrient source because the presence of grass seedlings in soil results in up to a 10-fold increase of PCL1444 cells. Moreover, the root colonized by strain PCL1444 was able to penetrate through an agar layer, resulting in the degradation of naphthalene underneath this layer. In addition, the inoculation of grass seeds or seedlings with PCL1444 protected them against naphthalene phytotoxicity. Finally, this plant-microbe combination appeared able to degrade naphthalene from soil that was heavily polluted with a complex mixture of polycyclic aromatic hydrocarbons. To our knowledge, this is the first time that a naturally occurring bacterium has been selected for the combination of the abilities to degrade a pollutant and colonize plant roots. We suggest that the principle described here, to select a bacterium which combines efficient root colonization with a beneficial activity, also can be used to improve the selection of other more efficient plant-bacterium pairs for beneficial purposes such as biocontrol, biofertilization, and phytostimulation.