Pseudomonas putida and its close relatives: mixing and mastering the perfect tune for plants.

Plant growth-promoting rhizobacteria (PGPR) are a group of microorganisms of utmost interest in agricultural biotechnology for their stimulatory and protective effects on plants. Among the various PGPR species, some Pseudomonas putida strains combine outstanding traits such as phytohormone synthesis, nutrient solubilization, adaptation to different stress conditions, and excellent root colonization ability. In this review, we summarize the state of the art and the most relevant findings related to P. putida and its close relatives as PGPR, and we have compiled a detailed list of P. putida sensu stricto, sensu lato, and close relative strains that have been studied for their plant growth-promoting characteristics. However, the mere in vitro analysis of these characteristics does not guarantee correct plant performance under in vivo or field conditions. Therefore, the importance of studying adhesion and survival in the rhizosphere, as well as responses to environmental factors, is emphasized. Although numerous strains of this species have shown good performance in field trials, their use in commercial products is still very limited. Thus, we also analyze the opportunities and challenges related to the formulation and application of bioproducts based on these bacteria. KEY POINTS: •The mini-review updates the knowledge on Pseudomonas putida as a PGPR. • Some rhizosphere strains are able to improve plant growth under stress conditions. • The metabolic versatility of this species encourages the development of a bioproduct.

Impact of Soil Rhizobacteria Inoculation and Leaf-Chewing Insect Herbivory on Mentha piperita Leaf Secondary Metabolites.

Secondary metabolites commonly play important physiological roles in plants and can be modified quantitatively and qualitatively by exposure to biotic and abiotic interactions. Plant growth promoting rhizobacteria (PGPR) and herbivory induce systemic resistance. In the present study, we analyzed the induction of secondary metabolites in peppermint plants in response to chewing insect herbivory on PGPR-inoculated Mentha piperita plants. The secondary metabolites of M. piperita plants were increased when plants were inoculated with PGPR and also exposed to caterpillar herbivory. It was found that the total essential oil yield in inoculated plants with insect damage was ~2.6-fold higher than in controls. The yield was similar to that of plants either damaged by insects or inoculated, indicating that there was no synergism. The same trend was observed for phenolic compounds. In contrast, VOC emissions were significantly higher in plants infested by insects, independent of whether they were inoculated. Insect damaged plants had 5.5 times higher monoterpene emissions than control plants, and ~ 2-fold higher emissions than on PGPR-inoculated plants without insects. To gain a better understanding of how herbivory on PGPR-inoculated plants can cause an increase in secondary metabolites of peppermint, we examined changes in plant defense hormones in inoculated plants after herbivory. We found that the combination of both treatments increased the endogenous jasmonic and salicylic acid levels to the same extent as in plants only inoculated or only insect-damaged. Because different interactions can alter the phytochemistry of plants such as M. piperita, this topic is both ecologically and economically relevant.

Plant growth promotion by Pseudomonas putida KT2440 under saline stress: role of eptA.

New strategies to improve crop yield include the incorporation of plant growth-promoting bacteria in agricultural practices. The non-pathogenic bacterium Pseudomonas putida KT2440 is an excellent root colonizer of crops of agronomical importance and has been shown to activate the induced systemic resistance of plants in response to certain foliar pathogens. In this work, we have analyzed additional plant growth promotion features of this strain. We show it can tolerate high NaCl concentrations and determine how salinity influences traits such as the production of indole compounds, siderophore synthesis, and phosphate solubilization. Inoculation with P. putida KT2440 significantly improved seed germination and root and stem length of soybean and corn plants under saline conditions compared to uninoculated plants, whereas the effects were minor under non-saline conditions. Also, random transposon mutagenesis was used for preliminary identification of KT2440 genes involved in bacterial tolerance to saline stress. One of the obtained mutants was analyzed in detail. The disrupted gene encodes a predicted phosphoethanolamine-lipid A transferase (EptA), an enzyme described to be involved in the modification of lipid A during lipopolysaccharide (LPS) biosynthesis. This mutant showed changes in exopolysaccharide (EPS) production, low salinity tolerance, and reduced competitive fitness in the rhizosphere.

Genomic and metabolic differences between Pseudomonas putida populations inhabiting sugarcane rhizosphere or bulk soil.

Pseudomonas putida is one of 13 major groups of Pseudomonas spp. and contains numerous species occupying diverse niches and performing many functions such as plant growth promotion and bioremediation. Here we compared a set of 19 P. putida isolates obtained from sugarcane rhizosphere or bulk soil using a population genomics approach aiming to assess genomic and metabolic differences between populations from these habitats. Phylogenomics placed rhizosphere versus bulk soil strains in separate clades clustering with different type strains of the P. putida group. Multivariate analyses indicated that the rhizosphere and bulk soil isolates form distinct populations. Comparative genomics identified several genetic functions (GO-terms) significantly different between populations, including some exclusively present in the rhizosphere or bulk soil strains, such as D-galactonic acid catabolism and cellulose biosynthesis, respectively. The metabolic profiles of rhizosphere and bulk soil populations analyzed by Biolog Ecoplates also differ significantly, most notably by the higher oxidation of D-galactonic/D-galacturonic acid by the rhizosphere population. Accordingly, D-galactonate catabolism operon (dgo) was present in all rhizosphere isolates and absent in the bulk soil population. This study showed that sugarcane rhizosphere and bulk soil harbor different populations of P. putida and identified genes and functions potentially associated with their soil niches.

Desiccation-induced viable but nonculturable state in Pseudomonas putida KT2440, a survival strategy.

The potential of Pseudomonas putida KT2440 to act as a plant-growth promoter or as a bioremediator of toxic compounds can be affected by desiccation. In the present work, the bacterial survival ratio (BSR) in response to air desiccation was evaluated for P. putida KT2440 in the presence of different protectors. The BSR in the presence of nonreducing disaccharides, such as trehalose, was high after 15 days of desiccation stress (occurring at 30°C and 50% relative humidity), whereas in the absence of a protector the bacterial counts diminished to nondetectable numbers (ca 2.8 log CFU/mL). The LIVE/DEAD staining method showed that bacteria protected with trehalose maintained increased numbers of green cells after desiccation while cells without protection were all observed to be red. This indicated that nonprotected bacteria had compromised membrane integrity. However, when nonprotected bacteria subjected to 18 days of desiccation stress were rehydrated for a short time with maize root exudates or for 48 h with water (prolonged rehydration), the bacterial counts were as high as that observed for those not subjected to desiccation stress, suggesting that the cells entered the viable but nonculturable (VBNC) state under desiccation and that they returned to a culturable state after those means of rehydration. Interestingly an increase in the green color intensity of cells that returned to a culturable state was observed using LIVE/DEAD staining method, indicating an improvement in their membrane integrity. Cellular activity in the VBNC state was determined. A GFP-tagged P. putida strain expressing GFP constitutively was subjected to desiccation. After 12 days of desiccation, the GFP-tagged strain lost culturability, but it exhibited active GFP expression, which in turn made the cells green. Furthermore, the expression of 16S rRNA, rpoN (housekeeping), mutL, mutS (encoding proteins from the mismatch repair complex), and oprH (encoding an outer membrane protein) were examined by RT-PCR. All evaluated genes were expressed by both types of cells, culturable and nonculturable, indicating active molecular processes during the VBNC state.

Impact of phosphate solubilizing bacteria on wheat (Triticum aestivum ) in the presence of pesticides.

Three phosphate solubilizing bacteria were isolated and identified by 16S rRNA sequencing as Pseudomonas putida, Pseudomonas sp and Pseudomonas fulva . The strains were subjected to plant biochemical testing and all the PGPR attributes were checked in the presence of pesticides (chlorpyrifos and pyriproxyfen). The phosphate solubilizing index of strain Ros2 was highest in NBRIP medium i.e 2.23 mm. All the strains showed acidic pH (ranges from 2.5-5) on both medium i.e PVK and NBRIP. Strain Ros2 was highly positive for ammonia production as well as siderophore production while strain Rad2 was positive for HCN production. The results obtained by the strains Rad1, Rad2 and Ros2 for auxin production were 33.1, 30.67 and 15.38 µg ml-1, respectively. Strain Rad1 showed 16% increase in percentage germination in comparison to control in the presence of pesticide stress. Most promising results for chlorophyll content estimation were obtained in the presence of carotenoids upto 6 mgg-1 without stress by both strains Rad1 and Rad2. Study suggests that especially strain Ros2 can enhance plant growth parameters in the pesticide stress.

Impact of phosphate solubilizing bacteria on wheat (Triticum aestivum ) in the presence of pesticides / Impacto das bactérias solubilizantes de fosfato no trigo (Triticum aestivum ) na presença de pesticidas

Abstract Three phosphate solubilizing bacteria were isolated and identified by 16S rRNA sequencing as Pseudomonas putida, Pseudomonas sp and Pseudomonas fulva . The strains were subjected to plant biochemical testing and all the PGPR attributes were checked in the presence of pesticides (chlorpyrifos and pyriproxyfen). The phosphate solubilizing index of strain Ros2 was highest in NBRIP medium i.e 2.23 mm. All the strains showed acidic pH (ranges from 2.5-5) on both medium i.e PVK and NBRIP. Strain Ros2 was highly positive for ammonia production as well as siderophore production while strain Rad2 was positive for HCN production. The results obtained by the strains Rad1, Rad2 and Ros2 for auxin production were 33.1, 30.67 and 15.38 µg ml-1, respectively. Strain Rad1 showed 16% increase in percentage germination in comparison to control in the presence of pesticide stress. Most promising results for chlorophyll content estimation were obtained in the presence of carotenoids upto 6 mgg-1 without stress by both strains Rad1 and Rad2. Study suggests that especially strain Ros2 can enhance plant growth parameters in the pesticide stress.

Resumo Três bactérias solubilizantes de fosfato foram isoladas e identificadas por seqüenciamento de rRNA 16S como Pseudomonas putida, Pseudomonas sp e Pseudomonas fulva. As estirpes foram submetidas a testes bioquímicos de plantas e todos os atributos PGPR foram verificados na presença de pesticidas (clorpirifos e piriproxifeno). O índice de solubilização de fosfato da estirpe Ros2 foi mais elevado no meio NBRIP, isto é, 2,23 mm. Todas as estirpes apresentaram um pH ácido (varia de 2,5-5) em ambos os meios, isto é PVK e NBRIP. A estirpe Ros2 foi altamente positiva para a produção de amoníaco, bem como a produção de sideróforos enquanto a estirpe Rad2 foi positiva para a produção de HCN. Os resultados obtidos pelas estirpes Rad1, Rad2 e Ros2 para a produção de auxina foram 33,1, 30,67 e 15,38 μg ml-1 , respectivamente. A deformação Rad1 mostrou aumento de 16% na germinação percentual em comparação com o controlo na presença de stress de pesticida. Os resultados mais promissores para a estimativa do teor de clorofila foram obtidos na presença de carotenóides até 6 mgg-1 sem estresse por ambas as cepas Rad1 e Rad2. Estudo sugere que especialmente a estirpe Ros2 pode melhorar parâmetros de crescimento de plantas no estresse de pesticidas.

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.

Compatible bacterial mixture, tolerant to desiccation, improves maize plant growth.

Plant growth-promoting rhizobacteria (PGPR) increase plant growth and crop productivity. The inoculation of plants with a bacterial mixture (consortium) apparently provides greater benefits to plant growth than inoculation with a single bacterial strain. In the present work, a bacterial consortium was formulated containing four compatible and desiccation-tolerant strains with potential as PGPR. The formulation had one moderately (Pseudomonas putida KT2440) and three highly desiccation-tolerant (Sphingomonas sp. OF178, Azospirillum brasilense Sp7 and Acinetobacter sp. EMM02) strains. The four bacterial strains were able to adhere to seeds and colonize the rhizosphere of plants when applied in both mono-inoculation and multi-inoculation treatments, showing that they can also coexist without antagonistic effects in association with plants. The effects of the bacterial consortium on the growth of blue maize were evaluated. Seeds inoculated with either individual bacterial strains or the bacterial consortium were subjected to two experimental conditions before sowing: normal hydration or desiccation. In general, inoculation with the bacterial consortium increased the shoot and root dry weight, plant height and plant diameter compared to the non-inoculated control or mono-inoculation treatments. The bacterial consortium formulated in this work had greater benefits for blue maize plants even when the inoculated seeds underwent desiccation stress before germination, making this formulation attractive for future field applications.

Potential of Pseudomonas putida PCI2 for the Protection of Tomato Plants Against Fungal Pathogens.

Tomato is one of the most economically attractive vegetable crops due to its high yields. Diseases cause significant losses in tomato production worldwide. We carried out Polymerase Chain Reaction studies to detect the presence of genes encoding antifungal compounds in the DNA of Pseudomonas putida strain PCI2. We also used liquid chromatography-electrospray tandem mass spectrometry to detect and quantify the production of compounds that increase the resistance of plants to diseases from culture supernatants of PCI2. In addition, we investigated the presence of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase in PCI2. Finally, PCI2 was used for inoculation of tomato seeds to study its potential biocontrol activity against Fusarium oxysporum MR193. The obtained results showed that no fragments for the encoding genes of hydrogen cyanide, pyoluteorin, 2,4-diacetylphloroglucinol, pyrrolnitrin, or phenazine-1-carboxylic acid were amplified from the DNA of PCI2. On the other hand, PCI2 produced salicylic acid and jasmonic acid in Luria-Bertani medium and grew in a culture medium containing ACC as the sole nitrogen source. We observed a reduction in disease incidence from 53.33 % in the pathogen control to 30 % in tomato plants pre-inoculated with PCI2 as well as increases in shoot and root dry weights in inoculated plants, as compared to the pathogenicity control. This study suggests that inoculation of tomato seeds with P. putida PCI2 increases the resistance of plants to root rot caused by F. oxysporum and that PCI2 produces compounds that may be involved at different levels in increasing such resistance. Thus, PCI2 could represent a non-contaminating management strategy potentially applicable in vegetable crops such as tomato.

Pseudomonas putida Stimulates Primordia on Agaricus bitorquis.

Casing layer is one step of Agaricus bisporus cultivation where there is a competitive environment with a high number of microorganisms and diversity interacting with mycelia. It is suggested that a minimal community of these microorganisms would be necessary to stimulate fructification. However, A. bisporus is not able to produce primordia in sterile casing layers or Petri dishes. Thus, the objective of this study was to characterize bacterial microbiota of casing layers from A. bisporus cultivation, isolate, identify and characterize the bacteria responsible for the stimulation of primordium and their action mechanism using Agaricus bitorquis as a primordium stimulation model. Bacterial and Pseudomonas spp. communities of different casing layers of A. bisporus cultivation were collected and quantified. It was concluded that Pseudomonas spp. corresponds to 75-85% of bacterial population of the casing layers in A. bisporus cultivation and among those 12% are Pseudomonas putida. Four biochemical assays were used to identify P. putida. In vitro primordium stimulation of living P. putida and non-living bacterial suspensions, after chemical or physical treatments, was tested using A. bitorquis as a primordium stimulation model. Primordium stimulation assay was registered by photographs, and micrographs of vertical cut of primordium were registered by scanning electron microscope. Interaction of living P. putida with A. bitorquis mycelia is capable of stimulating primordial instead of non-living bacterial suspensions. Stimulation of A. bitorquis primordia does not imply or is related to mycelial growth inhibition, but a hierarchical relation of primordium succession and development is suggested.

Anatomical, morphological, and phytochemical effects of inoculation with plant growth- promoting rhizobacteria on peppermint (Mentha piperita).

Plant growth-promoting rhizobacteria (PGPR) generally exert their effects through enhancement of plant nutrient status and/or phytohormone production. The effects of PGPR on aromatic plant species are poorly known. We measured plant growth parameters, chlorophyll content, trichome density, stomatal density, and levels of secondary metabolites in peppermint (Mentha piperita) seedlings inoculated with PGPR strains Bacillus subtilis GB03, Pseudomonas fluorescens WCS417r, P. putida SJ04, or a combination of WCS417r + SJ04. The treated plants, in comparison with controls, showed increases in shoot biomass, root biomass, leaf area, node number, trichome density, and stomatal density, and marked qualitative and quantitative changes in monoterpene content. Improved knowledge of the factors that control or affect biosynthesis of secondary metabolites and monoterpene accumulation will lead to strategies for improved cultivation and productivity of aromatic plants and other agricultural crops without the use of chemical fertilizers or pesticides.

Coordinated response of phospholipids and acyl components of membrane lipids in Pseudomonas putida A (ATCC 12633) under stress caused by cationic surfactants.

The present study assessed the role of membrane components of Pseudomonas putida A (ATCC 12633) under chemical stress conditions originated by treatment with tetradecyltrimethylammonium bromide (TTAB), a cationic surfactant. We examined changes in fatty acid composition and in the fluidity of the membranes of cells exposed to TTAB at a specific point of growth as well as of cells growing with TTAB. The addition of 10-50 mg TTAB l(-1) promoted an increase in the saturated/unsaturated fatty acid ratio. By using fluorescence polarization techniques, we found that TTAB exerted a fluidizing effect on P. putida A (ATCC 12633) membranes. However, a complete reversal of induced membrane fluidification was detected after 15 min of incubation with TTAB. Consistently, the proportion of unsaturated fatty acids was lower in TTAB-treated cells as compared with non-treated cells. In the presence of TTAB, the content of phosphatidylglycerol increased (120 %), whilst that of cardiolipin decreased (60 %). Analysis of the fatty acid composition of P. putida A (ATCC 12633) showed that phosphatidylglycerol carried the major proportion of saturated fatty acids (89 %), whilst cardiolipin carried an elevated proportion of unsaturated fatty acids (18 %). The increase in phosphatidylglycerol and consequently in saturated fatty acids, together with a decrease in cardiolipin content, enabled greater membrane resistance, reversing the fluidizing effect of TTAB. Therefore, results obtained in the present study point to changes in the fatty acid profile as an adaptive response of P. putida A (ATCC 12633) cells to stress caused by a cationic surfactant.

Effect of Pseudomonas putida on growth and anthocyanin pigment in two poinsettia (Euphorbia pulcherrima) cultivars.

Pseudomonas putida is plant growth promoting rhizobacteria (PGPR) that have the capacity to improve growth in plants. The purpose of this study was to determine growth and anthocyanin pigmentation of the bracts in two poinsettia Euphorbia pulcherrima cultivars (Prestige and Sonora Marble) using three strains of P. putida, as well as a mixture of the three (MIX). Comparison with the control group indicated for the most part that Prestige grew better than the Sonora Marble cultivars with the PGPR strains. Prestige with the MIX strain grew better compared to control for the number of cyathia (83 versus 70.4), volume of roots (45 versus 3 cm(3)), number of leaves (78 versus 58), and area of leaf (1,788 versus 1,331 cm(2)), except for the number of flowers (8.8 versus 11.6). To the naked eye, coloration of plants appeared identical in color compared to the control group. For all plants with P. putida strains, there was less anthocyanin pigment, but biomass was always greater with PGPR strains. Nevertheless, to the naked eye, the coloration of the plants appeared identical in color compared to the control group. This is the first study reporting the positive effects of P. putida rhizobacteria treatments on growth of poinsettia cultivars.