Plant Growth-promoting Pseudomonas Putida Research Articles

Alginate-perlite encapsulated Pseudomonas putida A (ATCC 12633) cells: Preparation, characterization and potential use as plant inoculants

Microbial immobilization can be used to prepare encapsulated inoculants. Here, we characterize and describe the preparation of Ca-alginate-perlite microbeads loaded with cells of plant growth-promoting Pseudomonas putida A (ATCC 12633), for their future application as agricultural inoculants. The microbeads were prepared by dropwise addition of a CaCl2-paraffin emulsion mixture to an emulsion containing alginate 2% (w/v), perlite 0.1–0.4% (w/v) and bacterial suspension in 0.9% NaCl (1010 CFU/mL). For all perlite concentrations used, microbead size was 90–120 μm, the trapped population was 108 CFU/g microbeads and the increase in mechanical stability was proportional to perlite concentration. Microbeads containing 0.4% (w/v) perlite were able to release bacteria into the medium after 30 days of incubation. When we evaluated how P. putida A (ATCC 12633) entrapped in Ca-alginate-perlite (0.4% (w/v)) microbeads colonized the Arabidopsis thaliana rhizosphere, an increase in colonization over time was detected (from an initial 2.1 × 104 to 9.2 × 105 CFU/g soil after 21 days). With this treatment, growth promotion of A. thaliana occurred with an increase in the amount of proteins, and in root and leaf biomass. It was concluded that the microbeads could be applied as possible inoculants, since they provide protection and a controlled release of microorganisms into the rhizosphere.

Pseudomonas putida NBRIC19 provides protection to neighboring plant diversity from invasive weed Parthenium hysterophorus L. by altering soil microbial community

Parthenium hysterophorus L. (Parthenium) is an invasive weed species which is spreading worldwide affecting natural ecological systems, biodiversity, crop production and human health. The present study was conducted to evaluate the potential of plant growth promoting Pseudomonas putida NBRIC19 in detoxifying the phytotoxic effect of Parthenium. Significant increase in C/N ratio, macronutrients, and micronutrients was observed in P. putida NBRIC19-treated soil. P. putida NBRIC19 treatment of the soil provided protection to plant communities in Parthenium invaded area, as the species diversity had increased in the treatment as compared to non-bacterized soil. P. putida NBRIC19 treatment besides Parthenium, also succeeded in controlling other weed species like Commelina benghalensis and Cynodon dactylon. In addition to this, the impact of Parthenium was also studied on functional microbial diversity based on carbon source utilization pattern. It was observed that P. putida NBRIC19 treatment of soil had shifted the microflora in such a manner that utilization of toxic allelochemicals increased to lessen their phytotoxic effect. Taken together, these results suggest that soil treatment with P. putida NBRIC19 may be used as a promising biological control measure for controlling the phytotoxic effect of Parthenium and in protecting ecosystem integrity of neighboring plants in Parthenium invaded areas.

Effect of inoculation with a thermotolerant plant growth promoting Pseudomonas putida strain AKMP7 on growth of wheat (Triticum spp.) under heat stress

The present study was carried out to investigate the effect of plant growth promoting thermotolerant Pseudomonas putida strain AKMP7 on the growth of wheat plants to heat stress. The results indicated the superior performance by P. putida strain AKMP7 in improving survival and growth of wheat plants under heat stress. The bacterium significantly increased the root and shoot length, dry biomass, tiller, spike let and grain formation of wheat over uninoculated plants. Inoculation reduced membrane injury and the activity of several antioxidant enzymes such as SOD, APX and CAT under heat stress. Inoculation improved the levels of cellular metabolites like proline, chlorophyll, sugars, starch, amino acids, and proteins compared to uninoculated plants. Scanning electron microscopy studies confirmed the colonization of the organism on the root surface. This result suggests the possible role of microorganisms in mitigating adverse effects of climate changes on crop growth and may lead to development of microbe based climate- ready technology.

Functional, genetic and chemical characterization of biosurfactants produced by plant growth-promotingPseudomonas putida267

Plant growth-promoting Pseudomonas putida strain 267, originally isolated from the rhizosphere of black pepper, produces biosurfactants that cause lysis of zoospores of the oomycete pathogen Phytophthora capsici. The biosurfactants were characterized, the biosynthesis gene(s) partially identified, and their role in control of Phytophthora damping-off of cucumber evaluated. The biosurfactants were shown to lyse zoospores of Phy. capsici and inhibit growth of the fungal pathogens Botrytis cinerea and Rhizoctonia solani. In vitro assays further showed that the biosurfactants of strain 267 are essential in swarming motility and biofilm formation. In spite of the zoosporicidal activity, the biosurfactants did not play a significant role in control of Phytophthora damping-off of cucumber, since both wild type strain 267 and its biosurfactant-deficient mutant were equally effective, and addition of the biosurfactants did not provide control. Genetic characterization revealed that surfactant biosynthesis in strain 267 is governed by homologues of PsoA and PsoB, two nonribosomal peptide synthetases involved in production of the cyclic lipopeptides (CLPs) putisolvin I and II. The structural relatedness of the biosurfactants of strain 267 to putisolvins I and II was supported by LC-MS and MS-MS analyses. The biosurfactants produced by Ps. putida 267 were identified as putisolvin-like CLPs; they are essential in swarming motility and biofilm formation, and have zoosporicidal and antifungal activities. Strain 267 provides excellent biocontrol activity against Phytophthora damping-off of cucumber, but the lipopeptide surfactants are not involved in disease suppression. Pseudomonas putida 267 suppresses Phy. capsici damping-off of cucumber and provides a potential supplementary strategy to control this economically important oomycete pathogen. The putisolvin-like biosurfactants exhibit zoosporicidal and antifungal activities, yet they do not contribute to biocontrol of Phy. capsici and colonization of cucumber roots by Ps. putida 267. These results suggest that Ps. putida 267 employs other, yet uncharacterized, mechanisms to suppress Phy. capsici.

Plant growth-promoting Pseudomonas putida WCS358 produces and secretes four cyclic dipeptides: cross-talk with quorum sensing bacterial sensors.

The most universal cell-cell signaling mechanism in Gram-negative bacteria occurs via the production and response to a class of small diffusible molecules called N-acylhomoserine lactones (AHLs). This communication is called quorum sensing and is responsible for the regulation of several physiological processes and many virulence factors in pathogenic bacteria. The detection of these molecules has been rendered possible by the utilization of genetically engineered bacterial biosensors which respond to the presence of exogenously supplied AHLs. In this study, using diverse bacterial biosensors, several biosensor activating fractions were purified by organic extraction, HPLC and TLC of cell-free culture supernatants of plant growth-promoting Pseudomonas putida WCS358. Surprisingly, it was observed that the most abundant compounds in these fractions were cyclic dipeptides (diketopiperazines, DKPs), a rather novel finding in Gram-negative bacteria. The purification, characterization, chemical synthesis of four DKPs are reported and their possible role in cell-cell signaling is discussed.

Cloning and characterisation of the rpoS gene from plant growth-promoting Pseudomonas putida WCS358: RpoS is not involved in siderophore and homoserine lactone production

The rpoS gene which encodes a stationary phase sigma factor has been identified and characterised from the rhizosphere-colonising plant growth-promoting Pseudomonas putida strain WCS358. The predicted protein sequence has extensive homologies with the RpoS proteins form other bacteria, in particular with the RpoS sigma factors of the fluorescent pseudomonads. A genomic transposon insertion in the rpoS gene was constructed, these mutants were analysed for their ability to produce siderophore (iron-transport agent) and the autoinducer quorum-sensing molecules called homoserine lactones (AHL). It was determined that RpoS was not involved in the regulation of siderophore and AHL production, synthesis of these molecules is important for gene expression at stationary phase. P. putida WCS358 produces at least three different AHL molecules.

Characterization of type II protein secretion (xcp) genes in the plant growth-stimulating Pseudomonas putida, strain WCS358.

In Pseudomonas aeruginosa, the products of the xcp genes are required for the secretion of exoproteins across the outer membrane. Despite structural conservation of the Xcp components, secretion of exoproteins via the Xcp pathway is generally not found in heterologous organisms. To study the specificity of this protein secretion pathway, the xcp genes of another fluorescent pseudomonad, the plant growth-promoting Pseudomonas putida strain WCS358, were cloned and characterized. Nucleotide sequence analysis revealed the presence of at least five genes, i.e., xcpP, Q, R, S, and T, with homology to xcp genes of P. aeruginosa. Unlike the genetic organization in P. aeruginosa, where the xcp cluster consists of two divergently transcribed operons, the xcp genes in P. putida are all oriented in the same direction, and probably comprise a single operon. Upstream of xcpP in P. putida, an additional open reading frame, with no homolog in P. aeruginosa, was identified, which possibly encodes a lipoprotein. Mutational inactivation of xcp genes in P. putida did not affect secretion, indicating that no proteins are secreted via the Xcp system under the growth conditions tested, and that an alternative secretion system is operative. To obtain some insight into the secretory pathway involved, the amino acid sequence of the N-terminus of the major extracellular protein was determined. The protein could be identified as flagellin. Mutations in the xcpQ and R genes of P. aeruginosa could not be complemented by introduction of the corresponding xcp genes of P. putida. However, expression of a hybrid XcpR protein, composed of the N-terminal one-third of P. aeruginosa XcpR and the C-terminal two-thirds of P. putida XcpR, did restore protein secretion in a P. aeruginosa xcpR mutant.

Influence of pseudobactin 358 on the iron nutrition of barley

The influence of pseudobactin 358 (PSB358) on the iron uptake and chlorophyll synthesis of hydroponically-grown barley seedlings ( Hordeum vulgare L. cv. Femina) was studied. PSB358 is the siderophore produced by the plant-growth promoting Pseudomonas putida strain WCS358. Iron uptake by iron-deficient barley plants was significantly higher with 59FePSB358 than with 59FeEDDHA as an iron source. However, iron uptake from the ferrated barley phytosiderophores ( 59FePS) was the most efficient. Chlorophyll synthesis in barley was stimulated by 4 μM FePSB358 compared with that in barley supplied with 4 μM FeCl 3. The unferrated PSB358 (4 μM) positively influenced chlorophyll synthesis in barley if the plants were grown with Fe(OH) 3 as an iron source, whereas 4 μM EDDHA had a negative effect. Under axenic conditions, chlorophyll synthesis was high and did not differ between plants that were grown with either FePSB358 or FeCl 3 as iron source. Inoculation of the axenic plants with barley-rhizosphere microorganisms reduced their chlorophyll synthesis and the phytosiderophore accumulation in their nutrient solution. Under these conditions, FePSB358 stimulated chlorophyll synthesis in barley compared with that in barley supplied with FeCl 3. It is concluded that both FePSB358 and PSB358 can affect the iron nutrition of barley positively, if, due to microbial degradation of the phytosiderophores, phytosiderophoremediated iron nutrition was not sufficient for the plant's iron requirement.

Siderophore receptor PupA as a marker to monitor wild-type Pseudomonas putida WCS358 in natural environments.

For application of genetically engineered fluorescent Pseudomonas spp., specific markers are required for monitoring of wild-type Pseudomonas strains and their genetically modified derivatives in natural environments. In this study, the specific siderophore receptor PupA of plant growth-promoting Pseudomonas putida WCS358 was used as a marker to monitor wild-type strain WCS358. After introduction into natural soil and rhizosphere environments, strain WCS358 could be recovered efficiently on a medium amended with 300 microM pseudobactin 358. Although low population densisties of indigenous pseudomonads (less than or equal to 10(3)/g of soil or root) were recovered on the pseudobactin 358-amended medium, subsequent agglutination assays with a WCS358-specific polyclonal antiserum enabled accurate monitoring of populations of wild-type strain WCS358 over a range of approximately 10(3) to 10(7) CFU/g of soil or root. Genetic analysis of the background population by PCR and Southern hybridization revealed that natural occurrence of the pupA gene was limited to a very small number of indigenous Pseudomonas spp. which are very closely related to P. putida WCS358. The PupA marker system enabled the study of differences in rhizosphere colonization among wild-type strain WCS358, rifampin-resistant derivative WCS358rr, and Tn5 mutant WCS358::xylE. Chromosomally mediated rifampin resistance did not affect the colonizing ability of P. putida WCS358. However, Tn5 mutant WCS358::xylE colonized the radish rhizosphere significantly less than did its parental strain.

Characterization of type IV pilus genes in plant growth-promoting Pseudomonas putida WCS358

In a search for factors that could contribute to the ability of the plant growth-stimulating Pseudomonas putida WCS358 to colonize plant roots, the organism was analyzed for the presence of genes required for pilus biosynthesis. The pilD gene of Pseudomonas aeruginosa, which has also been designated xcpA, is involved in protein secretion and in the biogenesis of type IV pili. It encodes a peptidase that processes the precursors of the pilin subunits and of several components of the secretion apparatus. Prepilin processing activity could be demonstrated in P. putida WCS358, suggesting that this nonpathogenic strain may contain type IV pili as well. A DNA fragment containing the pilD (xcpA) gene of P. putida was cloned and found to complement a pilD (xcpA) mutation in P. aeruginosa. Nucleotide sequencing revealed, next to the pilD (xcpA) gene, the presence of two additional genes, pilA and pilC, that are highly homologous to genes involved in the biogenesis of type IV pili. The pilA gene encodes the pilin subunit, and pilC is an accessory gene, required for the assembly of the subunits into pili. In comparison with the pil gene cluster in P. aeruginosa, a gene homologous to pilB is lacking in the P. putida gene cluster. Pili were not detected on the cell surface of P. putida itself, not even when pilA was expressed from the tac promoter on a plasmid, indicating that not all the genes required for pilus biogenesis were expressed under the conditions tested. Expression of pilA of P. putida in P. aeruginosa resulted in the production of pili containing P. putida PilA subunits.

Identification and characterization of a siderophore regulatory gene (pfrA) of Pseudomonas putida WCS358: homology to the alginate regulatory gene aigQ of Pseudomonas aeruginosa

Genes encoding biosynthesis of pseudobactin 358 (a microbial iron transport agent) and its cognate outer membrane receptor protein, PupA, are transcribed only under iron limitation in plant growth-promoting Pseudomonas putida WCS358. Two cosmid clones were identified from a gene bank of WCS358 DNA which could independently and in an iron-dependent manner activate transcription from a WCS358 siderophore gene promoter in heterologous Pseudomonas strain A225. The functional region of one of the clones was localized by subcloning, transposon Tn3Gus mutagenesis, and DNA sequencing. Genomic transposon insertion mutants in the functional region lost the capacity to activate a siderophore gene promoter fusion transcriptionally; furthermore, these mutants no longer produced pseudobactin 358. The activating region consisted of a single gene designated pfrA (Pseudomonas ferric regulator). The pfrA gene codes for a single polypeptide, PfrA, of approximately 18 kDa, which has 58% identity to AlgQ (also known as AlgR2), a positive regulator involved in transcriptionally regulating alginate biosynthesis in Pseudomonas aeruginosa. Cross-complementation studies between the pfrA gene of P. putida and the algQ gene of P. aeruginosa revealed that pfrA can restore mucoidy (alginate production) in an algQ mutant and that algQ could poorly complement a pfrA genomic mutant. It is concluded that PfrA is involved in the positive regulation of siderophore biosynthetic genes in response to iron limitation; furthermore, pfrA and algQ appeared to be interchangeable between P. putida and P. aeruginosa.

Stability of rifampicin resistance as a marker for root colonization studies of Pseudomonas putida in the field

The stability of rifampicin resistance in plant growth-promoting Pseudomonas putida strain WCS358 was studied in potato rhizosphere in the field. Three out of seven rifampicin-resistant mutants of strain WCS358 were selected in this study. Their specific growth rate, competitive growth in liquid medium and colonization of potato roots in non-sterile soil, was comparable to that of their parental strain. These rifampicin-resistant mutants were used to treat potato seed tubers, which were thereafter sown in the field. To test the stability of the rifampicin resistance in the field, about 1200 fluorescent Pseudomonas isolates obtained from underground plant parts at 82, 95, 109 and 130 days after seeding, were tested for rifampicin resistance and for agglutination with an antiserum specific for strain WCS358. Ail fluorescent Pseudomonas isolates that showed a positive agglutination reaction with the antiserum, were also rifampicin-resistant. Twelve agglutination-positive isolates, selected at random, were all identified as strain WCS358 from patterns of lipopolysaccharides after sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Thus, rifampicin resistance seems to be a stable marker in the mutants of strain WCS358 tested, also under field conditions. It is concluded that rifampicin resistance can be used as a reliable marker for ecological studies on rhizosphere pseudomonads.

Molecular analysis of iron transport in plant growth-promotingPseudomonas putida WCS358

Root-colonizingPseudomonas putida WCS358 enhances growth of potato in part by producing under iron-limiting conditions a yellow-green, fluorescent siderophore designated pseudobactin 358. This siderophore efficiently complexes iron(III) in the rhizosphere, making it less available to certain endemic microorganisms, including phytopathogens, thus inhibiting their growth. At least 15 genes distributed over five gene clusters are required for the biosynthesis of pseudobactin 358. High-affinity iron(III) transport in strain WCS358 is initiated by an 86-kDa outer membrane receptor protein (PupA) which appears to be specific for ferric pseudobactin 358. PupA shares strong similarity with TonB-dependent receptor proteins ofEscherichia coli, which suggests a TonB-like protein in strain WCS358 is required for iron(III) transport. Strain WCS358 possesses a second uptake system for ferric pseudobactin 358 and structurally diverse ferric siderophores produced by other microorganisms. A second receptor gene (pupB) responsible for iron transport from pseudobactin BN7 or pseudobactin BN8 has been identified. The production of this and certain other ferric siderophore receptor proteins requires that strain WCS358 be grown in the presence of these siderophores. An apparent regulatory gene required for the expression ofpupB is located adjacent topupB. Two positive regulatory genes have been identified which can independently activate, under low-iron(III) conditions, transcription of genes coding for the biosynthesis of pseudobactin 358.