Thomas F. C. Chin-A-Woeng

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.

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.

Interactions in the tomato rhizosphere of two Pseudomonas biocontrol strains with the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici.

The fungus Fusarium oxysporum f. sp. radicis-lycopersici causes foot and root rot of tomato plants, which can be controlled by the bacteria Pseudomonas fluorescens WCS365 and P. chlororaphis PCL1391. Induced systemic resistance is thought to be involved in biocontrol by P. fluorescens WCS365. The antifungal metabolite phenazine-1-carboxamide (PCN), as well as efficient root colonization, are essential in the mechanism of biocontrol by P. chlororaphis PCL1391. To understand the effects of bacterial strains WCS365 and PCL1391 on the fungus in the tomato rhizosphere, microscopic analyses were performed using different autofluorescent proteins as markers. Tomato seedlings were inoculated with biocontrol bacteria and planted in an F. oxysporum f. sp. radicis-lycopersici-infested gnotobiotic sand system. Confocal laser scanning microscope analyses of the interactions in the tomato rhizosphere revealed that i) the microbes effectively compete for the same niche, and presumably also for root exudate nutrients; ii) the presence of either of the two bacteria negatively affects infection of the tomato root by the fungus; iii) both biocontrol bacteria colonize the hyphae extensively, which may represent a new mechanism in biocontrol by these pseudomonads; and iv) the production of PCN by P. chlororaphis PCL1391 negatively affects hyphal growth and branching, which presumably affects the colonization and infecting ability of the fungus.

Description of the Colonization of a Gnotobiotic Tomato Rhizosphere by Pseudomonas fluorescens Biocontrol Strain WCS365, Using Scanning Electron Microscopy

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 ≈10 6 CFU per cm of root near the root base to ≈10 2 to 10 3 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 microcolonies are the sites where the intracellular N-acyl-Lhomoserine 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. Additional keywords: microbiological control, rhizosphere colonization.

Introduction of the phzH gene of Pseudomonas chlororaphis PCL1391 extends the range of biocontrol ability of Phenazine-1-Carboxylic Acid-Producing pseudomonas spp. strains

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. Its biocontrol activity is mediated by the production of phenazine-1-carboxamide (PCN). In contrast, the take-all biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84, which produce phenazine-1-carboxylic acid (PCA), do not control this disease. To determine the role of the amide group in biocontrol, the PCN biosynthetic genes of strain PCL1391 were identified and characterized. Downstream of phzA through phzG, the novel phenazine biosynthetic gene phzH was identified and shown to be required for the presence of the 1-carboxamide group of PCN because a phzH mutant of strain PCL1391 accumulated PCA. The deduced PhzH protein shows homology with asparagine synthetases that belong to the class II glutamine amidotransferases, indicating that the conversion of PCA to PCN occurs via a transamidase reaction catalyzed by PhzH. Mutation of phzH caused loss of biocontrol activity, showing that the 1-carboxamide group of PCN is crucial for control of tomato foot and root rot. PCN production and biocontrol activity of the mutant were restored by complementing the phzH gene in trans. Moreover, transfer of phzH under control of the tac promoter to the PCA-producing biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84 enabled these strains to produce PCN instead of PCA and suppress tomato foot and root rot. Thus, we have shown, for what we believe is the first time, that the introduction of a single gene can efficiently extend the range of the biocontrol ability of bacterial strains.

Phenazine-1-Carboxamide Production in the Biocontrol Strain Pseudomonas chlororaphis PCL1391 Is Regulated by Multiple Factors Secreted into the Growth Medium

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. The production of phenazine-1-carboxamide (PCN) is crucial for this biocontrol activity. In vitro production of PCN is observed only at high-population densities, suggesting that production is under the regulation of quorum sensing. The main autoinducer molecule produced by PCL1391 was identified structurally as N-hexanoyl-L-homoserine lactone (C6-HSL). The two other autoinducers that were produced comigrate with N-butanoyl-L-homoserine lactone (C4-HSL) and N-octanoyl-L-homoserine lactone (C8-HSL). Two PCL1391 mutants lacking production of PCN were defective in the genes phzI and phzR, respectively, the nucleotide sequences of which were determined completely. Production of PCN by the phzI mutant could be complemented by the addition of exogenous synthetic C6-HSL, but not by C4-HSL, C8-HSL, or any other HSL tested. Expression analyses of Tn5luxAB reporter strains of phzI, phzR, and the phz biosynthetic operon clearly showed that phzI expression and PCN production is regulated by C6-HSL in a population density-dependent manner. The introduction of multiple copies of the regulatory genes phzI and phzR on various plasmids resulted in an increase of the production of HSLs, expression of the PCN biosynthetic operon, and consequently, PCN production, up to a sixfold increase in a copy-dependent manner. Surprisingly, our expression studies show that an additional, yet unidentified factor(s), which are neither PCN nor C4-HSL or C8-HSL, secreted into the growth medium of the overnight cultures, is involved in the positive regulation of phzI, and is able to induce PCN biosynthesis at low cell densities in a growing culture, resulting in an increase of PCN production.

The sss Colonization Gene of the Tomato-Fusarium oxysporum f. sp. radicis-lycopersici Biocontrol Strain Pseudomonas fluorescens WCS365 Can Improve Root Colonization of Other Wild-type Pseudomonas spp. Bacteria

We show that the disease tomato foot and root rot caused by the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici can be controlled by inoculation of seeds with cells of the efficient root colonizer Pseudomonas fluorescens WCS365, indicating that strain WCS365 is a biocontrol strain. The mechanism for disease suppression most likely is induced systemic resistance. P. fluorescens strain WCS365 and P. chlororaphis strain PCL1391, which acts through the production of the antibiotic phenazine-1-carboxamide, were differentially labeled using genes encoding autofluorescent proteins. Inoculation of seeds with a 1:1 mixture of these strains showed that, at the upper part of the root, the two cell types were present as microcolonies of either one or both cell types. Microcolonies at the lower root part were predominantly of one cell type. Mixed inoculation tended to improve biocontrol in comparison with single inoculations. In contrast to what was observed previously for strain PCL1391, mutations in various colonization genes, including sss, did not consistently decrease the biocontrol ability of strain WCS365. Multiple copies of the sss colonization gene in WCS365 improved neither colonization nor biocontrol by this strain. However, introduction of the sss-containing DNA fragment into the poor colonizer P. fluorescens WCS307 and into the good colonizer P. fluorescens F113 increased the competitive tomato root tip colonization ability of the latter strains 16- to 40-fold and 8- to 16-fold, respectively. These results show that improvement of the colonization ability of wild-type Pseudomonas strains by genetic engineering is a realistic goal.

Influence of environmental conditions on the production of phenazine-1-carboxamide by Pseudomonas chlororaphis PCL1391

Pseudomonas chlororaphis PCL1391 produces the secondary metabolite phenazine-1-carboxamide (PCN), which is an antifungal metabolite required for biocontrol activity of the strain. Identification of conditions involved in PCN production showed that some carbon sources and all amino acids tested promote PCN levels. Decreasing the pH from 7 to 6 or decreasing the growth temperature from 21 to 16 degrees C decreased PCN production dramatically. In contrast, growth at 1% oxygen as well as low magnesium concentrations increased PCN levels. Salt stress, low concentrations of ferric iron, phosphate, sulfate, and ammonium ions reduced PCN levels. Fusaric acid, a secondary metabolite produced by the soilborne Fusarium spp. fungi, also reduced PCN levels. Different nitrogen sources greatly influenced PCN levels. Analysis of autoinducer levels at conditions of high and low PCN production demonstrated that, under all tested conditions, PCN levels correlate with autoinducer levels, indicating that the regulation of PCN production by environmental factors takes place at or before autoinducer production. Moreover, the results show that autoinducer production not only is induced by a high optical density but also can be induced by certain environmental conditions. We discuss our findings in relation to the success of biocontrol in the field.

Biocontrol Traits of Pseudomonas spp. Are Regulated by Phase Variation

Of 214 Pseudomonas strains isolated from maize rhizosphere, 46 turned out to be antagonistic, of which 43 displayed clear colony phase variation. The latter strains formed both opaque and translucent colonies, designated as phase I and phase II, respectively. It appeared that important biocontrol traits, such as motility and the production of antifungal metabolites, proteases, lipases, chitinases, and biosurfactants, are correlated with phase I morphology and are absent in bacteria with phase II morphology. From a Tn5luxAB transposon library of Pseudomonas sp. strain PCL1171 phase I cells, two mutants exhibiting stable expression of phase II had insertions in gacS. A third mutant, which showed an increased colony phase variation frequency was mutated in mutS. Inoculation of wheat seeds with PCL1171 bacteria of phase I morphology resulted in efficient suppression of take-all disease, whereas disease suppression was absent with phase II bacteria. Neither the gacS nor the mutS mutant was able to suppress take-all, but biocontrol activity was restored after genetic complementation of these mutants. Furthermore, in a number of cases, complementation by gacS of wild-type phase II sectors to phase I phenotype could be shown. A PCL1171 phase I mutant defective in antagonistic activity appeared to have a mutation in a gene encoding a lipopeptide synthetase homologue and had lost its biocontrol activity, suggesting that biocontrol by strain PCL1171 is dependent on the production of a lipopeptide. Our results show that colony phase variation plays a regulatory role in biocontrol by Pseudomonas bacteria by influencing the expression of major biocontrol traits and that the gacS and mutS genes play a role in the colony phase variation process. Therefore phase variation not only plays a role in escaping animal defense but it also appears to play a much broader and vital role in the ecology of bacteria producing exoenzymes, antibiotics, and other secondary metabolites.