Rhizobacterium Pseudomonas Putida Research Articles

2-Aminoethylphosphonate utilization in Pseudomonas putida BIRD-1 is controlled by multiple master regulators.

SummaryBacteria possess various regulatory mechanisms to detect and coordinate a response to elemental nutrient limitation. In pseudomonads, the two‐component system regulators CbrAB, NtrBC and PhoBR, are responsible for regulating cellular response to carbon (C), nitrogen (N) and phosphorus (P) respectively. Phosphonates are reduced organophosphorus compounds produced by a broad range of biota and typified by a direct C‐P bond. Numerous pseudomonads can use the environmentally abundant phosphonate species 2‐aminoethylphosphonate (2AEP) as a source of C, N, or P, but only PhoBR has been shown to play a role in 2AEP utilization. On the other hand, utilization of 2AEP as a C and N source is considered substrate inducible. Here, using the plant‐growth‐promoting rhizobacterium Pseudomonas putida BIRD‐1 we present evidence that 2AEP utilization is under dual regulation and only occurs upon depletion of C, N, or P, controlled by CbrAB, NtrBC, or PhoBR respectively. However, the presence of 2AEP was necessary for full gene expression, i.e. expression was substrate inducible. Mutation of a LysR‐type regulator, termed AepR, upstream of the 2AEP transaminase‐phosphonatase system (PhnWX), confirmed this dual regulatory mechanism. To our knowledge, this is the first study identifying coordination between global stress response and substrate‐specific regulators in phosphonate metabolism.

A comparison of induced antiviral resistance by the phytoprotein CAP-34 and isolate P1f of the rhizobacterium Pseudomonas putida.

CAP-34 is a previously reported phytoprotein isolated from Clerodendrum aculeatum (syn. Volkameria aculeata), inducing systemic antiviral resistance against plant virus infection in susceptible plants. This paper compares the resistance inducing efficacy of CAP-34 and a rhizobacterial isolate P1f on tomato (systemic) and tobacco Xanthi-nc (hypersensitive), against tobacco mosaic virus (TMV). The PGPR isolate was identified as an isolate of Pseudomonas putida through molecular and biochemical characterization, and it exhibited PGPR traits such as production of auxin and siderophore. GC-MS examination of the volatiles produced by P1f included several that are implicated in antimicrobial activity, growth promotion and induced systemic resistance. Foliar treatment of tobacco plants with P1f and CAP-34 led to an induced antiviral state in hypersensitive tobacco that persisted for 5 and 3days, post-treatment, respectively, with a percent reduction in lesion number greater than 90. A higher accumulation of hydrogen peroxide and production of peroxidase enzyme was recorded in P1f-treated leaves, in comparison to those with CAP-34 treatment. The disease incidence in tomato plants treated with CAP-34 and P1f was 30 and 60 percent, respectively, 28dpi. A significant increase was noted in growth parameters such as number of branches and flowers in CAP-34 treated plants, while a significant enhancement in plant height and dry shoot and root weight was observed in P1f-treated set, compared to the control set. ELISA values for the presence of TMV were significantly lower in the infected tomato plants in the treated sets, as compared to the control set, with CAP-34 treatment exhibiting better results as against the P1f-treated set. In the resistant plants from either set, no viral RNA or viral coat protein was detected through RT-PCR and serology. These results suggest that CAP-34 affords more pronounced protection against virus infection compared to the rhizobacterial isolate P1f.

Alleviation of zinc deficiency in wheat inoculated with root endophytic fungus Piriformospora indica and rhizobacterium Pseudomonas putida

Plant growth, grain yield, and the nutritional grain quality reduce in Zinc (Zn)-deficient soils. As a practical approach, the utilization of plant growth-promoting microorganisms offers a promising approach to cope with Zinc deficiency. This study was carried out to determine the effects of endophytic fungus Piriformospora indica and rhizobacterium Pseudomonas putida on growth, nutrient status, and antioxidant enzyme activities of two wheat (Triticum aestivum L.) genotypes under Zn deficiency. The experiment was conducted using a completely randomized factorial design with three replications and three treatments: wheat genotypes [Zn-efficient (H-Zn) genotype, Azadi and Zn-inefficient (L-Zn) genotype, Niknejad]), Zn concentrations (0 and 2 μmol Zn L−1) and microbial inoculations (P. indica and P. putida). Under Zn deficiency, P. indica, P. putida, and dual inoculation of P. indica with P. putida significantly increased the shoot dry weight of L-Zn genotype up to 10.3%, 25.6% and, 15.4% compared to control plants, respectively. Inoculation of P. indica led to an increase in thinner and highly-branched roots and subsequently increased Zn uptake in the shoot of L-Zn genotype under Zn deficiency. Under such condition, individual inoculation of L-Zn genotype with P. putida and P. indica increased peroxidase activity by 85.7% and 40% compared with non-inoculated plants. Malondialdehyde content of both wheat genotypes inoculated with P. indica was significantly decreased compared to the non-inoculated plants. Generally, depending on the wheat genotype, P. indica and P. putida can increase plant tolerance by affecting only part of the tolerance mechanisms (including antioxidant and non-antioxidant mechanisms) under Zn deficiency stress. In summary, P. indica and P. putida could improve the fitness of the L-Zn genotype under Zn-deficiency stress.

Plant growth-stimulating rhizobacteria capable of producing L-amino acids.

Pseudomonas putida BIRD-1 is a microorganism that inhabits the rhizosphere and solubilizes phosphate and iron and produces indolacetic acid [Roca, A., Pizarro-Tobías, P., Udaondo, Z., Fernández, M., Matilla, M.A., Molina-Henares, M.A., et al. (2013) Analysis of plant growth-promoting properties encoded by the genome of the rhizobacterium Pseudomonas putida BIRD-1. Environ Microbiol 15: 780-794]. In this study, we generated mutant strains that are capable of producing the plant growth stimulating compounds L-tryptophan and L-phenylalanine. We prepared clones that overproduce L-tryptophan by first mutagenizing P. putida BIRD-1, then by selecting for clones in the presence of inhibitory concentrations of 5-fluoro-D,L-tryptophan. The production of this aromatic amino acid was confirmed by chemical analysis and cross-feeding experiments with auxotrophs. One of the mutants, named P. putida BIRD-1-12, was mutagenized again to isolate clones that are also able to grow in the presence of inhibitory concentrations of p-fluoro-D,L-phenylalanine. One of these resulting clones was then isolated and named BIRD-1-12F. Our analysis revealed that the strains that either overproduce L-tryptophan, or L-tryptophan and L-phenylalanine, excel at promoting the growth of a number of plant crops of agricultural interest.

Effects of ACC deaminase containing rhizobacteria on plant growth and expression of Toc GTPases in tomato (Solanum lycopersicum) under salt stress

The bacterial enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase plays a key role in lowering plant stress ethylene levels, thereby stimulating plant growth. The present study aims to evaluate whether the ACC deaminase producing plant growth promoting rhizobacterium (PGPR) Pseudomonas putida UW4 can maintain and promote plant growth in saline environments and modulate the expression of chloroplast import apparatus genes in salt-treated tomato plants (Solanum lycopersicum L.). Tomatoes were grown in the presence and absence of the PGPR and shoot length, fresh and dry mass, and chlorophyll concentration were measured after 6 weeks. The expression levels of the Toc GTPases of the chloroplast protein import apparatus were measured using quantitative real-time PCR. The results showed that the rhizobacteria significantly increased shoot length, shoot fresh and dry mass, and the chlorophyll concentration of tomato seedlings grown in the presence of up to 90 mmol·L–1NaCl. Analysis showed that the expression of most of the Toc GTPase genes was upregulated in tomato seedlings after 6 weeks of exposure to NaCl, which may help facilitate the import into chloroplasts of proteins that are involved in the stress response.

Responses of unsaturated Pseudomonas putida CZ1 biofilms to environmental stresses in relation to the EPS composition and surface morphology.

The extracellular polymeric substance (EPS) and surface properties of unsaturated biofilms of a heavy metal-resistant rhizobacterium Pseudomonas putida CZ1, in response to aging, pH, temperature and osmotic stress, were studied by quantitative analysis of EPS and atomic force microscope. It was found that EPS production increased approximately linearly with culture time, cells in the air-biofilm interface enhanced EPS production and decreased cell volume to cope with nutrient depletion during aging. Low pH, high temperature and certain osmotic stress (120 mM NaCl) distinctly stimulated EPS production, and the main component enhanced was extracellular protein. In addition to the enhancement of EPS production in response to high osmotic (328 mM NaCl) stress, cells in the biofilm adhere tightly together to maintain a particular microenvironment. These results indicated the variation of EPS composition and the cooperation of cells in the biofilms is important for the survival of Pseudomonas putida CZ1 from environmental stresses in the unsaturated environments such as rhizosphere.

Reprogramming of fatty acid and oxylipin synthesis in rhizobacteria-induced systemic resistance in tomato

The rhizobacterium Pseudomonas putida BTP1 stimulates induced systemic resistance (ISR) in tomato. A previous work showed that the resistance is associated in leaves with the induction of the first enzyme of the oxylipin pathway, the lipoxygenase (LOX), leading to a faster accumulation of its product, the free 13-hydroperoxy octadecatrienoic acid (13-HPOT), 2days after Botrytis cinerea inoculation. In the present study, we further investigated the stimulation of the oxylipin pathway: metabolites and enzymes of the pathway were analyzed to understand the fate of the 13-HPOT in ISR. Actually the stimulation began upstream the LOX: free linolenic acid accumulated faster in P. putida BTP1-treated plants than in control. Downstream, the LOX products 13-fatty acid hydroperoxides esterified to galactolipids and phospholipids were more abundant in bacterized plants than in control before infection. These metabolites could constitute a pool that will be used after pathogen attack to produce free fungitoxic metabolites through the action of phospholipase A2, which is enhanced in bacterized plants upon infection. Enzymatic branches which can use as substrate the fatty acid hydroperoxides were differentially regulated in bacterized plants in comparison to control plants, so as to lead to the accumulation of the most fungitoxic compounds against B. cinerea. Our study, which is the first to demonstrate the accumulation of an esterified defense metabolite during rhizobacteria-mediated induced systemic resistance, showed that the oxylipin pathway is differentially regulated. It suggests that this allows the plant to prepare to a future infection, and to respond faster and in a more effective way to B. cinerea invasion.

Effect of fungicides on plant growth promoting activities of phosphate solubilizing Pseudomonasputida isolated from mustard (Brassica compestris) rhizosphere

This study was navigated to examine the effects of fungicide-stress on the activities of plant-growth-promoting rhizobacterium Pseudomonasputida with inherent phosphate solubilizing activity. The fungicide-tolerant and phosphate solubilizing P.putida strain PS9 was isolated from the mustard rhizosphere and tentatively identified following standard morphological, physiological and biochemical tests. To further consolidate the identity of the strain PS9, the 16S rDNA sequence analysis was performed. Following the BLAST program, the strain PS9 was identified as P.putida. In the presence of the varying concentrations (0–3200μgmL−1; at a two fold dilution interval) of four fungicides of different chemical families (tebuconazole, hexaconazole, metalaxyl and kitazin) amended in minimal salt agar medium, the P.putida strain PS9 showed a variable tolerance levels (1400–3200μgmL−1) against the tested fungicides. The strain PS9 produced plant-growth-promoting (PGP) substances in significant amount in the absence of fungicides. In general, fungicides applied at the recommended, two and three times of the recommended rates, decreased the PGP attributes of P.putida the strain PS9 and affected the PGP activities in concentration-dependent manner. Fungicides at the recommended dose had minor reducing effect while the doses higher than the recommended dose significantly reduced the PGP activities (phosphate solubilization, salicylic acid, 2,3-dihydroxy benzoic acid, and indole-3-acetic acid production except exo-polysaccharides, hydrogen cyanate and ammonia production). Of the four fungicides, tebuconazole generally, showed maximum toxicity to the PGP activities of the strain PS9. This study inferred that fungicides must be examined in vitro for their possible adverse effects on soil micro flora before their application in agricultural fields. Moreover, the results also suggested the prerequisite of application of fungicide-tolerant PGPR strains as bioinoculants so that their PGP activities may not be suppressed under fungicide stress.

Assessment of Plant Growth Promoting Activities of Rhizobacterium Pseudomonas putida under Insecticide-Stress

Assessment of Plant Growth Promoting Activities of Rhizobacterium Pseudomonas putida under Insecticide-Stress

Effects of organic wastes,Glomus intraradices andPseudomonas putida on the growth of tomato and on the reproduction of the Root-knot nematodeMeloidogyne incognita

Effects of organic wastes (biosolids, horse manure, sawdust and neem leaf litter [NLL]), an arbuscular mycorrhizal (AM) fungusGlomus intraradices, and a plant growth-promoting rhizobacteriumPseudomonas putida, were studied on the growth of tomato and on the reproduction ofMeloidogvne incognita. Pseudomonas putida andG. intraradices promoted tomato growth in nematode-infected and nematode-free plants but growth promotion was higher in the infected ones. WhenP. putida andG. intraradices were applied together, the increase in tomato growth was greater than when either agent was applied alone. Of the organic wastes, NLL was better in improving tomato growth of nematode-infected plants followed by biosolids, horse manure and sawdust. Combined use of NLL withP. putida plusG. intraradices was best in improving growth of the infected plants. Root colonization byP. putida was increased more when inoculated withG. intraradices than when inoculated singly. Of the organic wastes, use of sawdust withP. putida caused a greater increase in root colonization by fluorescent pseudomonads followed by NLL, horse manure and biosolids. Nematode parasitism had an adverse effect on root colonization byP. putida. Inoculation ofP. putida and organic wastes increased the root colonization caused by the AM fungus.P. putida was better in reducing galling and nematode multiplication thanG. intraradices, whereas use of the two together was better than that of either of them alone. Among organic wastes, NLL was better in reducing galling and nematode multiplication followed by biosolids, horse manure and sawdust. Combined use of NLL withP. putida plusG. intraradices was better in reducing galling and nematode multiplication than any other treatment.

Synergistic effects of antagonistic fungi and a plant growth promoting rhizobacterium, an arbuscular mycorrhizal fungus, or composted cow manure on populations of Meloidogyne incognita and growth of tomato

Effects of four antagonistic fungi (Paecilomyces lilacinus, Pochonia chlamydosporia, Trichoderma harzianum and Gliocladium virens) alone and together with a plant growth promoting rhizobacterium Pseudomonas putida, an arbuscular mycorrhizal fungus Glomus intraradices or with composted cow manure (CCM) were assessed on the growth of tomato and on the reproduction of Meloidogyne incognita in glasshouse experiments. Application of all antagonistic fungi (except G. virens), P. putida, G. intraradices or CCM caused a significant increase in the growth of plants without nematodes. However, use of either of these fungi, P. putida, G. intraradices and CCM against plants with nematodes caused a significant increase in tomato growth. Paecilomyces lilacinus caused a 42% increase in the growth of nematode-inoculated plants followed by P. chlamydosporia (36%), T. harzianum (18%) and G. virens (15%). CCM caused about 57% increase in the growth of nematode-inoculated plants followed by P. putida (37%) and G. intraradices (31%). Maximum increase (71%) in the growth of nematode-inoculated plants was observed when CCM was used with P. lilacinus. Moreover, P. lilacinus caused a high reduction (55%) in galling and nematode multiplication, while G. virens the least (25%). Use of P. putida also caused a 39% reduction in galling and nematode multiplication followed by CCM (34%) and G. intraradices (32%). Combined use of CCM with P. lilacinus caused maximum reduction (79%) in galling and nematode multiplication. Re-isolation of antagonistic fungi from nematodes revealed that P. lilacinus parasitised more females and eggs than other antagonistic fungi. Root colonisation by P. putida was increased with P. lilacinus, while colonisation by G. intraradices was reduced in the presence of antagonistic fungi.

Expression and characterization of 1-aminocyclopropane-1-carboxylate deaminase from the rhizobacterium Pseudomonas putida UW4: a key enzyme in bacterial plant growth promotion

The enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACCD) converts ACC, the precursor of the plant hormone ethylene, to α-ketobutyrate and ammonium. This enzyme has been identified in soil bacteria and has been proposed to play a key role in microbe-plant association. A soluble recombinant ACCD from Pseudomonas putida UW4 of molecular weight 41 kDa has been cloned, expressed, and purified. It showed selectivity and high activity towards the substrate ACC: K M=3.4±0.2 mM and k cat=146±5 min -1 at pH 8.0 and 22 °C. The enzyme displayed optimal activity at pH 8.0 with a sharp decline to essentially no activity below pH 6.5 and a slightly less severe tapering in activity at higher pH resulting in loss of activity at pH>10. The major component of the enzyme's secondary structure was determined to be α-helical by circular dichroism (CD). P. putida UW4 ACCD unfolded at 60 °C as determined by its CD temperature profile as well as by differential scanning microcalorimetry (DSC). Enzyme activity was knocked out in the point mutant Gly44Asp. Modeling this mutation into the known yeast ACCD structure shed light on the role this highly conserved residue plays in allowing substrate accessibility to the active site. This enzyme's biochemical and biophysical properties will serve as an important reference point to which newly isolated ACC deaminases from other organisms can be compared.

Plant-dependent active biological containment system for recombinant rhizobacteria.

This work describes the construction of the rhizobacterium Pseudomonas putida strain CS-4, which contains an active biological containment (ABC) system that ensures bacterial suicide in the absence of proline. Maize plants exudate proline, which allows CS-4 to colonize the rhizosphere at a similar level to that of the wild-type strain. However, when the plants are removed, the CS-4 population decreased in bulk soil at a higher rate than the wild-type strain.

Relationship between antifreeze protein and freezing resistance in Pseudomonas putida GR12-2.

Following transposon Tn5 mutagenesis of the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2, mutants that have different freeze-resistant properties were selected. Five of the freeze-sensitive mutants, i.e. FSM-5, -6, -14, -29, and -41, secreted a lower amount of antifreeze protein-(AFP) into the culture broth compared with the wild-type. Among of these five mutants, the three mutants (FSM-6, FSM-14, and FSM-41) that have the lowest level of freezing resistance (4.0-6.0% survival) also produce AFP at low levels (0.5-0.9 microg/mL) compared with the wild-type (4.8 microg/ml). The antifreeze and ice-nucleating activities of the AFP from these three mutant strains were similar to those of wild-type. Furthermore, the decreased freezing resistance from three mutants could be partially restored by adding purified AFP to mutant cell suspensions. Freezing resistance of three mutants was found to increase in proportion to the addition of AFP up to a concentration of 50 microg/mL. We conclude that accumulation of AFP is one component of the mechanism for freezing resistance in bacteria.

Effect of Wild-Type and Mutant Plant Growth-Promoting Rhizobacteria on the Rooting of Mung Bean Cuttings.

Mung bean cuttings were dipped in solutions of wild type and mutant forms of the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2 and then incubated for several days until roots formed. The bacteria P. putida GR12-2 and P. putida GR12-2/aux1 mutant do not produce detectable levels of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, whereas P. putida GR12-2/acd36 is an ACC deaminase minus mutant. All bacteria produce the phytohormone indole-3-acetic acid (IAA), and P. putida GR12-2/aux1 overproduces it. Treatment of cuttings with the above-mentioned bacteria affected the rates of ethylene production in the cuttings in a way that can be explained by the combined effects of the activity of ACC deaminase localized in the bacteria and bacterial produced IAA. P. putida GR12-2 and P. putida GR12-2/acd36-treated cuttings had a significantly higher number of roots compared with cuttings rooted in water. In addition, the wild type influenced the development of longer roots. P. putida GR12-2/aux1 stimulated the highest rates of ethylene production but did not influence the number of roots. These results are consistent with the notion that ethylene is involved in the initiation and elongation of adventitious roots in mung bean cuttings.

Isolation and characterization of an antifreeze protein with ice nucleation activity from the plant growth promoting rhizobacteriumPseudomonas putidaGR12-2

An antifreeze protein secreted to the growth medium by the plant growth promoting rhizobacterium Pseudomonas putida GR12-2 was purified to apparent homogeneity. The purified protein has a molecular mass of 164 ± 15 kDa and an isoelectric point of 5.3, contains both carbohydrate and lipid moieties, and is relatively rich in glycine and alanine. The properties of the purified antifreeze protein are similar to the properties previously reported for bacterial ice-nucleation proteins. In fact, the purified antifreeze protein also displays a low level of ice-nucleation activity. Removal of approximately 92 kDa of carbohydrate from the 164-kDa antifreeze glycoprotein did not noticeably alter the antifreeze activity of the molecule, although it did diminish the ice-nucleation activity. This is the first report of an antifreeze protein that also is active as an ice-nucleation protein.Key words: antifreeze protein, plant growth promoting rhizobacteria, freezing tolerance, ice-nucleation protein.

Early development of canola seedlings in the presence of the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2

The early development of seedlings from canola seeds treated with MgSO 4, P. putida GR12-2 or its mutant P. putida GR12-2/ acd68 lacking ACC deaminase activity (seed inoculation), or the growth of seedlings planted in soil containing one of these treatments (soil inoculation) was assessed. Root and shoot lengths, fresh and dry wt, and the chlorophyll and protein contents of shoots were monitored in unstressed control seedlings and in seedlings growing in a saline soil or exposed to cold night temperatures. Under all conditions including salt or temperature stress, the wild-type bacterium, but not the mutant, promoted root growth, and in those experiments in which the treatment was added to the soil, the wild-type bacterium promoted shoot growth.

Isolation and Characterization of Mutants of the Plant Growth-Promoting Rhizobacterium Pseudomonas putida GR12-2 That Overproduce Indoleacetic Acid

Following transposon Tn5 mutagenesis of the plant growth-promoting rhizobacterium Pseudomonas putida GR12-2, mutants that were able to grow in the presence of the tryptophan analog 5-fluorotryptophan were selected. Seven of the 50 5-fluorotryptophan-resistant mutants overproduced the phytohormone indoleacetic acid (IAA). Of these seven mutants, the highest level of IAA was observed with strain P. putida GR12-2/aux1, which produced four times the amount of indoleacetic acid synthesized by the wild-type strain. Strain P. putida GR12-2/aux1, in contrast to the wild type, lost the ability to stimulate the elongation of the roots of canola seedlings under gnotobiotic conditions. The growth rate, siderophore production, and 1-aminocyclopropane-1-carboxylate deaminase activity of mutant strain P. putida GR12-2/aux1 were identical to those of the wild-type strain. The role of IAA in the mechanism of plant growth stimulation by P. putida GR12-2 and other plant growth-promoting rhizobacteria is discussed.

Low temperature growth, freezing survival, and production of antifreeze protein by the plant growth promoting rhizobacterium Pseudomonas putida GR12-2.

The plant growth promoting rhizobacterium Pseudomonas putida GR12-2 was originally isolated from the rhizosphere of plants growing in the Canadian High Arctic. Here we report that this bacterium was able to grow and promote root elongation of both spring and winter canola at 5 degrees C, a temperature at which only a relatively small number of bacteria are able to proliferate and function. In addition, the bacterium survived exposure to freezing temperatures, i.e., -20 and -50 degrees C. In an effort to determine the mechanistic basis for this behaviour, it was discovered that following growth at 5 degrees C, P. putida GR12-2 synthesized and secreted to the growth medium a protein with antifreeze activity. Analysis of the spent growth medium, following concentration by ultrafiltration, by SDS-polyacrylamide gel electrophoresis revealed the presence of one major protein with a molecular mass of approximately 32-34 kDa and a number of minor proteins. However, at this point it is not known which of these proteins contains the antifreeze activity.

Overcoming the metabolic load associated with the presence of plasmid DNA in the plant growth promoting rhizobacteriumPseudomonas putidaGR12-2

When the broad host range plasmid vector pGSS15 was used to genetically transform the plant growth promoting rhizobacterium Pseudomonas putida GR12-2, the transformants were physiologically debilitated. It was postulated that the expression of the β-lactamase gene of pGSS15 caused a metabolic load resulting in the impaired functioning of the bacterium. To test this hypothesis, derivatives of pGSS15 that either lack the β-lactamase gene (pYH122) or in which a β-glucosidase gene was substituted for the β-lactamase gene (pYH124) were constructed and examined to see whether their presence also impaired the functioning of P. putida GR12-2. On the basis of growth rates, siderophore production, and the ability to stimulate canola root elongation in sterile growth pouches, neither of the newly constructed plasmids debilitated P. putida GR12-2. In addition, P. putida GR12-2 transformed with pYH124 facilitated the proliferation of the bacterium in minimal medium containing cellobiose at low temperature. This latter trait may enable P. putida GR12-2 to persist in the soil in competition with other microorganisms.Key words: plant growth promoting rhizobacteria, PGPR, bacterial fertilizer, soil bacteria, metabolic load, β-glucosidase