Mineral Phosphate Solubilization Research Articles

Regulation of Pyrroloquinoline Quinone-Dependent Glucose Dehydrogenase Activity in the Model Rhizosphere-Dwelling Bacterium Pseudomonas putida KT2440.

Soil-dwelling microbes solubilize mineral phosphates by secreting gluconic acid, which is produced from glucose by a periplasmic glucose dehydrogenase (GDH) that requires pyrroloquinoline quinone (PQQ) as a redox coenzyme. While GDH-dependent phosphate solubilization has been observed in numerous bacteria, little is known concerning the mechanism by which this process is regulated. Here we use the model rhizosphere-dwelling bacterium Pseudomonas putida KT2440 to explore GDH activity and PQQ synthesis, as well as gene expression of the GDH-encoding gene (gcd) and PQQ biosynthesis genes (pqq operon) while under different growth conditions. We also use reverse transcription-PCR to identify transcripts from the pqq operon to more accurately map the operon structure. GDH specific activity and PQQ levels vary according to growth condition, with the highest levels of both occurring when glucose is used as the sole carbon source and under conditions of low soluble phosphate. Under these conditions, however, PQQ levels limit in vitro phosphate solubilization. GDH specific activity data correlate well with gcd gene expression data, and the levels of expression of the pqqF and pqqB genes mirror the levels of PQQ synthesized, suggesting that one or both of these genes may serve to modulate PQQ levels according to the growth conditions. The pqq gene cluster (pqqFABCDEG) encodes at least two independent transcripts, and expression of the pqqF gene appears to be under the control of an independent promoter and terminator. Plant growth promotion can be enhanced by soil- and rhizosphere-dwelling bacteria by a number of different methods. One method is by promoting nutrient acquisition from soil. Phosphorus is an essential nutrient that plants obtain through soil, but in many cases it is locked up in forms that are not available for plant uptake. Bacteria such as the model bacterium Pseudomonas putida KT2440 can solubilize insoluble soil phosphates by secreting gluconic acid. This chemical is produced from glucose by the activity of the bacterial enzyme glucose dehydrogenase, which requires a coenzyme called PQQ. Here we have studied how the glucose dehydrogenase enzyme and the PQQ coenzyme are regulated according to differences in bacterial growth conditions. We determined that glucose dehydrogenase activity and PQQ production are optimal under conditions when the bacterium is grown with glucose as the sole carbon source and under conditions of low soluble phosphate.

Plant growth-promoting Burkholderia species isolated from annual ryegrass in Portuguese soils.

To search for culturable Burkholderia species associated with annual ryegrass in soils from natural pastures in Portugal, with plant growth-promoting effects. Annual ryegrass seedlings were used to trap Burkholderia from two different soils in laboratory conditions. A combined approach using genomic fingerprinting and sequencing of 16S rRNA and recA genes resulted in the identification of Burkholderia strains belonging to the species Burkholderia graminis, Burkholderia fungorum and the Burkholderia cepacia complex. Most strains were able to solubilize mineral phosphate and to synthesize indole acetic acid; some of them could produce siderophores and antagonize the phytopathogenic oomycete, Phytophthora cinnamomi. A strain (G2Bd5) of B.graminis was selected for gnotobiotic plant inoculation experiments. The main effects were the stimulation of root growth and enhancement of leaf lipid synthesis and turnover. Fluorescence insitu hybridization and confocal laser microscopy evidenced that strain G2Bd5 is a rhizospheric and endophytic colonizer of annual ryegrass. This work revealed that annual ryegrass can naturally associate with members of the genus Burkholderia. A novel plant growth promoting strain of B.graminis was obtained. The novel strain belongs to the plant-associated Burkholderia cluster and is a promising candidate for exploitation as plant inoculant in field conditions.

Mineral Phosphate Solubilizing Bacteria Isolated from Various Plant Rhizosphere under Different Aluminum Content

The objectives of this study was to isolate and characterize the mineral phosphate solubilizing bacteriafrom rhizosphere and evaluate their potential as plant growth promoting bacteria in Al-toxic soils. The halozone formation method was used to isolate PSB using the media containing insoluble phosphates (Ca-P or Al-P)as a source of phosphate. Eight of acid and Al-tolerant PSB isolates that were able to solubilize Ca-P wereobtained from rhizosphere of clover, wheat, corn, and sunflower grown in Al-toxic soil. Identification of theisolates based on the 16S rRNA gene sequence analysis demonstrated that the isolates were strains of Burkholderia(5 strains), Pseudomonas (1 strain), Ralstonia (1 strain), and unidentified bacterium (1 strains). All PSB isolatesshowed the capability to dissolve Ca-P, and only 1 strain (Ralstonia strain) was able to dissolve Al-P in agar platemedium. The P-solubilization by these isolates was correlated with pH of medium. Inoculation of the bacterialstrains on clover on Al-toxic medium showed that all isolates increased the plant dry weight compared withuninoculated treatment. Our results showed that those PSB isolates have potential to be developed as a biofertilizerto increase the efficiency of P-inorganic fertilizer used in Al-toxic soils.

Derepression of Mineral Phosphate Solubilization Phenotype by Insertional Inactivation of iclR in Klebsiella pneumoniae.

The mode of succinate mediated repression of mineral phosphate solubilization and the role of repressor in suppressing phosphate solubilization phenotype of two free-living nitrogen fixing Klebsiella pneumoniae strains was studied. Organic acid mediated mineral phosphate solubilization phenotype of oxalic acid producing Klebsiella pneumoniae SM6 and SM11 were transcriptionally repressed by IclR in presence of succinate as carbon source. Oxalic acid production and expression of genes of the glyoxylate shunt (aceBAK) was found only in glucose but not in succinate- and glucose+succinate-grown cells. IclR, repressor of aceBAK operon, was inactivated using an allelic exchange system resulting in derepressed mineral phosphate solubilization phenotype through constitutive expression of the glyoxylate shunt. Insertional inactivation of iclR resulted in increased activity of the glyoxylate shunt enzymes even in succinate-grown cells. An augmented phosphate solubilization up to 54 and 59% soluble phosphate release was attained in glucose+succinate-grown SM6Δ and SM11Δ strains respectively, compared to glucose-grown cells, whereas phosphate solubilization was absent or negligible in wildtype cells grown in glucose+succinate. Both wildtype and iclR deletion strains showed similar indole-3-acetic acid production. Wheat seeds inoculated with wildtype SM6 and SM11 improved both root and shoot length by 1.2 fold. However, iclR deletion SM6Δ and SM11Δ strains increased root and shoot length by 1.5 and 1.4 folds, respectively, compared to uninoculated controls. The repressor inactivated phosphate solubilizers better served the purpose of constitutive phosphate solubilization in pot experiments, where presence of other carbon sources (e.g., succinate) might repress mineral phosphate solubilization phenotype of wildtype strains.

Solubilization of inorganic phosphate by Pseudomonas strains isolated from rice rhizosphere

The use of phosphate solubilizing bacteria to mobilize large reserves of insoluble P in soils instead of phosphate fertilizers is an ecologically safe and economically reasonable alternative to assure a sustainable agriculture. In this work, the mineral phosphate solubilization activity was evaluated for 4 Pseudomonas strains isolated from rice rhizosphere. For qualitative tests, different media were used; NBRIP, YED and Pikovskaya (PVK) amended with 0.5% Ca3(PO4)2 and modified PVK medium containing CaHPO4, Ca5HO13P3, FePO4 and AlPO4 as P source. All bacteria were able to dissolve calcium phosphates, while none was surrounded by transparent halos when FePO4 and AlPO4 were the P source. Quantitative test was assessed in PVK substituting glucose by sucrose and galactose. The highest levels of soluble P were recorded in the presence of glucose, while it didn’t exceed 43.64 μg / ml and 19.19 µg / ml in the presence of galactose and sucrose, respectively. Therefore, glucose has been chosen as a carbon source to assay the effect of carbon concentration on P solubilization ability. The level of dissolved P was enhanced with increasing amount of glucose (1%, 2% and 3% (w/v)). In all cases, the P biosolubilisation was accompanied by a significant pH decrease of the liquid media. A significant inverse correlation was found between P concentrations and pH reduction. Thus, acidification of the media seems to be the main mechanism for calcium phosphates solubilization in this study, and these test strains could serve as efficient biofertilizer candidates for improving crop production in low P soils.

Biodiversity and plant growth promoting traits of culturable endophytic actinobacteria associated with Jatropha curcas L. growing in Panxi dry-hot valley soil

One of the proposed mechanisms through which plant growth-promoting endophyte (PGPE) enhances plant growth is the production of 1-aminocyclopropane-1-carboxylic acid deaminase (ACCD). However, information about the endophytic actinobacteria with ACC deaminase activity associated with native plants is still very scarce. In this study, a total of 257 endophytic actinobacterial isolates were obtained using actinobacteria-selective media from surface sterilized roots, stems, leaves and seeds of the oil-seed plant Jatropha curcas L. collected from dry-hot valley soil. Morphological and the 16S rRNA sequence analysis showed that most of the isolates belong to the Streptomyces genus and other non-Streptomyces strains distributed onto 13 genera, with several new species. 19 strains were found to have ACC deaminase activity and they belong to the genera Streptomyces, Nonomuraea, Micrococcus and Kibdelosporangium. The functional ability of the ACC deaminase producing isolates to produce indole-3-acetic acid (IAA), siderophores, mineral phosphate solubilization and growth on nitrogen free semi-solid medium was also determined. Seven strains were selected to inoculate the axenically grown seedlings and they resulted in a significant increase in the seedling fresh weight, the seedling length, the root length and the leaf area of the endophytes-treated seedlings compared to the control. This is the first report on the diversity and characterization of endophytic actinobacteria associated with important oil-seed plant J. curcas L. Our results demonstrate that some endophytic actinobacterial strains have the promising PGP attributes to be developed as biofertilizers to enhance soil fertility and promote the plant growth.

Rhizobium Mutant Deficient in Mineral Phosphate Solubilization Activity Shows Reduced Nodulation and Plant Growth in Green Gram

The aim of the present study was to understand the mineral phosphate solubilization (MPS) in Rhizobium sp. MR-54 (MPS+) by using forward genetic approach and its effect on nodulation as well as nitrogen accumulation on green gram. Tn5 mutants (MPS−) were generated from the MPS+ strain by Tn5 mutagenesis. Screening 1,603 colonies of MR-54::Tn5 resulted in identification of those mutants which failed to show any solubilization zone up to 72 h after incubation on Tri-calcium phosphate medium (TCP). pH drops and Pi release were highly correlated up to 15 days after incubation in TCP broth. Gluconic acid was detected in the culture supernatants of MPS+ isolates through thin layer chromatography indicating the presence of direct oxidation pathway in MR-54 strain. Detailed characterization of mutants was carried out for related properties like colony morphology, Pi release and pH change in TCP broth, organic acid production, surface characteristics and plant growth promoting ability etc. Mutants deficient in MPS showed reduced nodulation and nitrogen accumulation in green gram under green house conditions in leonard jar apparatus over the wild strain indicating the MPS by Rhizobium being involved in nodulation, nitrogen accumulation and plant growth.

Mineral phosphate solubilization by Streptomyces sp. CTM396 involves the excretion of gluconic acid and is stimulated by humic acids.

The actinomycetes isolates (128) which were taken from agricultural soil samples and collected near a rock phosphate processing unit were screened for mineral phosphate-solubilizing (MPS) ability. A significant MPS activity was observed for 30 isolates on various phosphate sources when grown in the National Botanical Research Institute's phosphate broth. CTM396 and CTM397 strains which showed the highest MPS abilities were identified by 16S rDNA sequencing as members of the genus Streptomyces. Their MPS activity was proved to be concomitant with a drop in pH due to the secretion of gluconic acid (GA). This was correlated with the simultaneous detection by PCR of genes gdh [encoding the glucose dehydrogenase (GDH) responsible for GA production from glucose] and pqq (involved in biosynthesis of the pyrroloquinoline quinone cofactor of GDH), as well as the highlighting of GHD enzyme activity, for the first time in a Streptomyces sp. strain producing GA. Furthermore, the 0.05% of humic acids proved to have a stimulatory effect on the growth and the ability of CTM396 to solubilize Gafsa rock phosphate. According to this study, it is possible to use humic acids and Gafsa rock phosphate in association with spores of ad hoc Streptomyces strains as natural and efficient amendments to improve plant growth with no need of costly and pollutant transformation of Gafsa rock phosphate.

Isolation and Characterization of Phosphate solubilising Burkholderia spp from the crops rhizosphere

It is an important trait in plant growth-promoting bacteria the ability to solubilize and mineralize insoluble inorganic phosphate compounds, making the element available for plants. High populations of phosphate solubilizing bacteria (PSB) will increases the P uptake by plants and reduce the application of organic fertilizers. In this study, a total of 14 PSB isolates from rhizosphere of maize, cotton, ragi, rice, turmeric, sugarcane, cowpea and green gram were tested for the mineral phosphate solubilizing (MPS) activities in Hydroxy Apatite (HAP) broth by analyzing the soluble-P content after 72 h of incubation at 30 0C. The ‘P’ solubilising ability of SGN 1 was found to be 35.56 mg/100ml isolated from sugarcane rhizosphere soil. SGN 1 which was showing higher phosphate solubilizing ability in HAP broth was selected for molecular characterization with amplifying and sequencing of 1.3 kb 16S rRNA gene. Based on the BLASTn homology it was found that PSB sugarcane isolate SGN1 having 95% identity with showed 99% homology with Burkholderia thailandensis.

Disruption of gene pqqA or pqqB reduces plant growth promotion activity and biocontrol of crown gall disease by Rahnella aquatilis HX2.

Rahnella aquatilis strain HX2 has the ability to promote maize growth and suppress sunflower crown gall disease caused by Agrobacterium vitis, A. tumefaciens, and A. rhizogenes. Pyrroloquinoline quinone (PQQ), a cofactor of aldose and alcohol dehydrogenases, is required for the synthesis of an antibacterial substance, gluconic acid, by HX2. Mutants of HX2 unable to produce PQQ were obtained by in-frame deletion of either the pqqA or pqqB gene. In this study, we report the independent functions of pqqA and pqqB genes in relation to PQQ synthesis. Interestingly, both the pqqA and pqqB mutants of R. aquatilis eliminated the ability of strain HX2 to produce antibacterial substance, which in turn, reduced the effectiveness of the strain for biological control of sunflower crown gall disease. The mutation also resulted in decreased mineral phosphate solubilization by HX2, which reduced the efficacy of this strain as a biological fertilizer. These functions were restored by complementation with the wild-type pqq gene cluster. Additionally, the phenotypes of HX2 derivatives, including colony morphology, growth dynamic, and pH change of culture medium were impacted to different extents. Our findings suggested that pqqA and pqqB genes individually play important functions in PQQ biosynthesis and are required for antibacterial activity and phosphorous solubilization. These traits are essential for R. aquatilis efficacy as a biological control and plant growth promoting strain. This study enhances our fundamental understanding of the biosynthesis of an environmentally significant cofactor produced by a promising biocontrol and biological fertilizer strain.

Whole genome sequencing and analysis of plant growth promoting bacteria isolated from the rhizosphere of plantation crops coconut, cocoa and arecanut.

Coconut, cocoa and arecanut are commercial plantation crops that play a vital role in the Indian economy while sustaining the livelihood of more than 10 million Indians. According to 2012 Food and Agricultural organization's report, India is the third largest producer of coconut and it dominates the production of arecanut worldwide. In this study, three Plant Growth Promoting Rhizobacteria (PGPR) from coconut (CPCRI-1), cocoa (CPCRI-2) and arecanut (CPCRI-3) characterized for the PGP activities have been sequenced. The draft genome sizes were 4.7 Mb (56% GC), 5.9 Mb (63.6% GC) and 5.1 Mb (54.8% GB) for CPCRI-1, CPCRI-2, CPCRI-3, respectively. These genomes encoded 4056 (CPCRI-1), 4637 (CPCRI-2) and 4286 (CPCRI-3) protein-coding genes. Phylogenetic analysis revealed that both CPCRI-1 and CPCRI-3 belonged to Enterobacteriaceae family, while, CPCRI-2 was a Pseudomonadaceae family member. Functional annotation of the genes predicted that all three bacteria encoded genes needed for mineral phosphate solubilization, siderophores, acetoin, butanediol, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, chitinase, phenazine, 4-hydroxybenzoate, trehalose and quorum sensing molecules supportive of the plant growth promoting traits observed in the course of their isolation and characterization. Additionally, in all the three CPCRI PGPRs, we identified genes involved in synthesis of hydrogen sulfide (H2S), which recently has been proposed to aid plant growth. The PGPRs also carried genes for central carbohydrate metabolism indicating that the bacteria can efficiently utilize the root exudates and other organic materials as energy source. Genes for production of peroxidases, catalases and superoxide dismutases that confer resistance to oxidative stresses in plants were identified. Besides these, genes for heat shock tolerance, cold shock tolerance and glycine-betaine production that enable bacteria to survive abiotic stress were also identified.

Artificial citrate operon and Vitreoscilla hemoglobin gene enhanced mineral phosphate solubilizing ability of Enterobacter hormaechei DHRSS.

Mineral phosphate solubilization by bacteria is mediated through secretion of organic acids, among which citrate is one of the most effective. To overproduce citrate in bacterial systems, an artificial citrate operon comprising of genes encoding NADH-insensitive citrate synthase of E. coli and Salmonella typhimurium sodium-dependent citrate transporter was constructed. In order to improve its mineral phosphate solubilizing (MPS) ability, the citrate operon was incorporated into E. hormaechei DHRSS. The artificial citrate operon transformant secreted 7.2 mM citric acid whereas in the native strain, it was undetectable. The transformant released 0.82 mM phosphate in flask studies in buffered medium containing rock phosphate as sole P source. In fermenter studies, similar phenotype was observed under aerobic conditions. However, under microaerobic conditions, no citrate was detected and P release was not observed. Therefore, an artificial citrate gene cluster containing Vitreoscilla hemoglobin (vgb) gene under its native promoter, along with artificial citrate operon under constitutive tac promoter, was constructed and transformed into E. hormaechei DHRSS. This transformant secreted 9 mM citric acid under microaerobic conditions and released 1.0 mM P. Thus, incorporation of citrate operon along with vgb gene improves MPS ability of E. hormaechei DHRSS under buffered, microaerobic conditions mimicking rhizospheric environment.

Overexpression of citrate operon in Herbaspirillum seropedicae Z67 enhances organic acid secretion, mineral phosphate solubilization and growth promotion of Oryza sativa

Herbaspirillum seropedicae Z67, nitrogen fixing endophyte, significantly promotes the growth of cereals. Organic acid secreting nitrogen fixing rhizobacteria have better plant growth promotion abilities due to mineral phosphate solubilization. Plasmids pAB7, pJNK3 and pJNK4 containing Escherichia coli cs (gltA), NADH insensitive cs (gltA1), and citrate operon consisting of gltA1 gene along with Salmonella typhimurium Na+ dependent citrate transporter (citC) gene under constitutive lac promoter were constructed in broad host range plasmid pUCPM18-Kmr. The plasmid transformants of H. seropedicae Z67 were obtained by electroporation. Hs (pAB7) and Hs (pJNK3) had increased CS activity but citric acid secretion was not significant. Hs (pJNK3) secreted 45 mM acetic acid while Hs (pJNK4) secreted 2.7 mM citric and 51 mM acetic acids. Hs (pJNK3) and Hs (pJNK4) released 80 μM and 110 μM amount of P from rock phosphate, respectively, in buffered medium under both aerobic and micro aerobic conditions. These transformants showed better plant growth promoting factors. Upon inoculation to rice plants (Gujarat – 17), increase of Fresh weight, Dry weight N, P and K content was observed. Thus the study demonstrates that artificial citrate operon in H. seropedicae Z67 enhances phosphate solubilization and plant growth promotion abilities.

Mineral Phosphate Solubilization: Concepts and Prospects in Sustainable Agriculture

Mineral Phosphate Solubilization: Concepts and Prospects in Sustainable Agriculture

Pseudomonas fluorescens ATCC 13525 Containing an Artificial Oxalate Operon and Vitreoscilla Hemoglobin Secretes Oxalic Acid and Solubilizes Rock Phosphate in Acidic Alfisols

Oxalate secretion was achieved in Pseudomonas fluorescens ATCC 13525 by incorporation of genes encoding Aspergillus niger oxaloacetate acetyl hydrolase (oah), Fomitopsis plaustris oxalate transporter (FpOAR) and Vitreoscilla hemoglobin (vgb) in various combinations. Pf (pKCN2) transformant containing oah alone accumulated 19 mM oxalic acid intracellularly but secreted 1.2 mM. However, in the presence of an artificial oxalate operon containing oah and FpOAR genes in plasmid pKCN4, Pf (pKCN4) secreted 13.6 mM oxalate in the medium while 3.6 mM remained inside. This transformant solubilized 509 μM of phosphorus from rock phosphate in alfisol which is 4.5 fold higher than the Pf (pKCN2) transformant. Genomic integrants of P. fluorescens (Pf int1 and Pf int2) containing artificial oxalate operon (plac-FpOAR-oah) and artificial oxalate gene cluster (plac-FpOAR-oah, vgb, egfp) secreted 4.8 mM and 5.4 mM oxalic acid, released 329 μM and 351 μM P, respectively, in alfisol. The integrants showed enhanced root colonization, improved growth and increased P content of Vigna radiata plants. This study demonstrates oxalic acid secretion in P. fluorescens by incorporation of an artificial operon constituted of genes for oxalate synthesis and transport, which imparts mineral phosphate solubilizing ability to the organism leading to enhanced growth and P content of V. radiata in alfisol soil.

Inoculation of phosphate-solubilizing bacteria Bacillus thuringiensis B1 increases available phosphorus and growth of peanut in acidic soil

Phosphate-solubilizing microorganisms play an important role in plant nutrition by enhancing phosphorus (P) availability to roots through converting the insoluble phosphates into soluble ions. We isolated phosphate-solubilizing bacteria (PSB) from acidic soil (Ultisols) in the field from the layer of 0–150 mm at a tea garden located at 28°38′26″ N and 116°24′27″ E. The capacity of bacterial isolates to solubilize mineral phosphate was tested on aluminum phosphate (AlPO4) in liquid medium. Among these PSB, isolate B1 (identified as Bacillus thuringiensis) exhibited the maximum P-solubilizing ability and was particularly efficient at solubilizing AlPO4 (up to 321 mg L−1) in vitro. The isolate B1 was inoculated to an acidic soil to study its effect on phosphate solubilization and growth of peanuts (Arachis hypogeae). The Olsen-P in the tested soil increased from 14.7 to 23.4 mg kg−1, with solubilization of 16.4 mg kg−1 soil of Occluded-P after 14-day incubation. The inoculation by B1 significantly increased plant height (from 37.7 to 45.7 cm), number of branches (from 34.0 to 52.7 per plant), hundred-seed weight (from 42.1 to 46.9 g) and crude protein content (from 243.5 to 268.2 g kg−1 dry weight). The phosphate-solubilizing B. thuringiensis strain B1 showed potential as a biological phosphorus fertilizer.

Heterologous expression of pyrroloquinoline quinone (pqq) gene cluster confers mineral phosphate solubilization ability to Herbaspirillum seropedicae Z67

Gluconic acid secretion mediated by the direct oxidation of glucose by pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) is responsible for mineral phosphate solubilization in Gram-negative bacteria. Herbaspirillum seropedicae Z67 (ATCC 35892) genome encodes GDH apoprotein but lacks genes for the biosynthesis of its cofactor PQQ. In this study, pqqE of Erwinia herbicola (in plasmid pJNK1) and pqq gene clusters of Pseudomonas fluorescens B16 (pOK53) and Acinetobacter calcoaceticus (pSS2) were over-expressed in H. seropedicae Z67. Transformants Hs (pSS2) and Hs (pOK53) secreted micromolar levels of PQQ and attained high GDH activity leading to secretion of 33.46mM gluconic acid when grown on 50mM glucose while Hs (pJNK1) was ineffective. Hs (pJNK1) failed to solubilize rock phosphate, while Hs (pSS2) and Hs (pOK53) liberated 125.47μM and 168.07μM P, respectively, in minimal medium containing 50mM glucose under aerobic conditions. Moreover, under N-free minimal medium, Hs (pSS2) and Hs (pOK53) not only released significant P but also showed enhanced growth, biofilm formation, and exopolysaccharide (EPS) secretion. However, indole acetic acid (IAA) production was suppressed. Thus, the addition of the pqq gene cluster, but not pqqE alone, is sufficient for engineering phosphate solubilization in H. seropedicae Z67 without compromising growth under nitrogen-fixing conditions.

Nodulation competitiveness of Ensifer meliloti alfalfa Nodule Isolates and Their Potential for Application as Inoculants

Alfalfa (Medicago sativa) is a widely cultivated legume, which enters into nitrogen-fixing symbiosis with Ensifer (Sinorhizobium) spp. In this study, an autochthonous rhizobial population of Ensifer sp. occupying alfalfa nodules grown in arable soil was used as the basis for selection of potential inoculants. Alfalfa nodule isolates were identified as Ensifer meliloti by partial 16S rDNA, recA, atpD and nodC nucleotide sequencing. The sampled isolates displayed different symbiotic performance and diversity in the number of plasmids and molecular weight. Isolates that were the most efficient in symbiotic nitrogen fixation were tagged with a constitutively expressed gusA gene carried by a stable plasmid vector pJBA21Tc and used in competition experiments in soil under greenhouse conditions. Two E. meliloti strains LU09 and LU12, which effectively competed with indigenous soil rhizobia, were selected. The metabolic profiles of these selected strains showed differences in the use of carbon and energy sources. In addition, the LU09 strain exhibited bacteriocin production and LU12 mineral phosphate solubilization, which are valuable traits for soil survival. These strains may be considered as potential biofertilizers for alfalfa cultivation.

Diversity, mechanism and biotechnology of phosphate solubilising microorganism in mangrove—A review

Abstract Phosphorus (P) is one of the major essential macronutrients for biological growth and development of plants. Phosphorous in soil is mainly found as mineral phosphorous or organic phosphorous which is however insoluble and unavailable to the plants. Microorganisms, both bacteria and fungi play a central role in the natural phosphorus cycle and convert insoluble forms of phosphorus to an accessible form which is an important trait for the growth and survival of plants. Among the phosphate solubilizing microbes, strains from the bacterial genera Pseudomonas, Bacillus and Rhizobium and fungi such as Pencillium, Aspergillus, Fusarium, Helminthosparium, Alternaria, etc. are the most powerful phosphate solubilizers. Phosphorous solubilization by microorganisms is a complex phenomenon, which depends on many factors such as nutritional, physiological and growth condition of the culture. The principal mechanism for mineral phosphate solubilization is the production of organic acids where the enzyme phosphatases play a major role in the mineralization of organic phosphorous in soil. In recent years several phosphatases encoding genes have been cloned and characterized and a few genes involved in mineral phosphate solubilization have been isolated. Therefore, genetic manipulation for improvement of phosphate-solubilizing bacteria to improve plant growth may include cloning genes involved in both mineral and organic phosphate solubilization, followed by their expression in selected rhizobacterial strains is an interesting approach. Besides phosphate solubilizing activity of microorganisms, the present paper also reports biotechnological potentials of phosphate solubilizing microorganisms from mangrove environment which is a unique saline costal ecosystem of tropical and subtropical regions of the world.

Investigation on Phosphate Solubilization Potential of Agricultural Soil Bacteria as Affected by Different Phosphorus Sources, Temperature, Salt, and pH

Ten phosphate-solubilizing bacterial strains belonging to genera Pseudomonas, Burkholderia, Enterobacter, Serratia, Klebsiella, and Aeromonas were tested for mineral phosphate solubilization activity in Pikovskaya's broth using different phosphate sources at four temperatures (15, 25, 35, and 45 οC). Dicalcium and tricalcium phosphate were solubilized more effectively (≥1000 mg L−1) than ferric and rock phosphate (≥100 mg L−1) and 35 °C was found to be the optimum temperature. Although Klebsiella and Aeromonas spp. are well known for their dinitrogen (N2)–fixing ability, to the best of our knowledge, this is the first report of inorganic phosphate solubilization by Klebsiella terrigena and Aeromonas vaga. Interestingly, A. vaga BAM-77 is the most efficient strain at solubilizing inorganic phosphorus (P) even in the presence of 8% sodium chloride (NaCl) at pH 10. These findings indicate that all four strains are efficient P solubilizers under variable conditions of temperature, pH, and P source and thus can be recommended for P fertilization in different soils.