Pikovskaya Research Articles

Phosphorus recycling mediated by Pseudomonas aeruginosa from eutrophic biochar

Present study focused on Phosphorus (P) enriched i.e., eutrophic biochar and its solubilization utilizing Pseudomonas aeruginosa (NCIM 8650). Previously established processes for lesser toxic and P rich biochar production (T-B-400, T-B-600 and T-B-800) using phosphogypsum pond wastewater soaked banana peduncles at three different pyrolysis temperatures (400, 600 and 800 °C) were utilized for the experiments. Bacteria inoculated in biochar loaded Pikovskaya (PVK) broth (modified: 0.2 g biochar/100 ml) was analyzed for 15 days about P solubilization along with control (modified broth without Pseudomonas aeruginosa). Gradual increase of P solubilization was observed with maximum values for T-B-400 and T-B-600 on 9th day in variation to 11th day of T-B-800. Different organic acids (Carbamic, Propanoic and Stearic hydrazide) ascertained through GCMS analysis along with significant pH change (7 to 3.41, 4.1 and 3.26 for T-B-400, T-B-600 and T-B-800 respectively) played major role toward P solubilization of modified PVK broth and highlighted significantly (p < 0.05) high release of P from T-B-400 (76%) than T-B-600 (65%) and T-B-800 (71%) over control. The FE-SEM, FTIR and XRD analysis (9th day: T-B-400/T-B-600; 11th day: T-B-800) of biochar residues in modified broth showed distinct morphological, structural and mineralogical changes in comparison to control. The findings highlights the potential of P solubilization by Pseudomonas aeruginosa from euthophic biochar, and hence provide opportunities for nutrients recovery from wastes.

Isolation of Inorganic Phosphorus-Solubilizing Bacteria from the Rhizosphere of Festuca arundinacea Schreb.

Inorganic phosphorus-solubilizing bacteria promote plant growth. However, different strains have different effects. The objective of this study was to screen efficient inorganic phosphorus-solubilizing bacteria (iPSB) from the rhizosphere of Festuca arundinacea Schreb. using Pikovskaya (PVK) medium. The results showed that 43 iPSB isolates were screened by clear halo zone production on PVK medium. Among them, the phosphate solubilization index (PSI) on the PVK medium of 31 iPSBs was calculated to be more than 1.5 at 28 °C in 7 days and ranged from 1.52 to 2.63. The best 31 iPSB isolates were selected for further study, including molecular identification using the 16S rRNA gene, testing of their P solubilization capacity in PVK broth medium, and the detection of catalase activity and organic acid and siderophore production. Among the 31 isolates, the phosphate solubilization of 8 isolates ranged from 203.96 to 412.22 µg/mL, and the value was significantly larger than that of the PVK broth medium without any bacterial isolates. These isolates were identified and belonged to Acinetobacter calcoaceticus, Buttiauxella sp., and Erwinia piriflorinigrans by sequence analysis of the 16S rRNA gene. These iPSB bacterial isolates enrich plant growth-promoting rhizobacteria and can promote the development of sustainable agriculture as phosphate fertilizer synergists.

Phosphorus-Solubilizing Capacity of Mortierella Species Isolated from Rhizosphere Soil of a Poplar Plantation.

Phosphorus is one of the main nutrients necessary for plant growth and development. Phosphorus-dissolving microorganisms may convert insoluble phosphorus in soil into available phosphorus that plants can easily absorb and utilize. In this study, four phosphorus-solubilizing fungi (L3, L4, L5, and L12) were isolated from the rhizosphere soil of a poplar plantation in Dongtai, Jiangsu Province, China. Phylogenetic analysis based on the internal transcribed spacer (ITS) and large subunit (LSU) of the ribosomal DNA sequences showed that the ITS and 28S sequences of isolates were the most similar to those of Mortierella. Morphological observation showed that most colonies grew in concentric circles and produced spores under different culture conditions. These results and further microscopic observations showed that these isolated fungi belonged to the genus Mortierella. Pikovskaya (PKO) medium, in which tricalcium phosphate was the sole phosphorus source, was used to screen strain L4 with the best phosphorus-solubilizing effect for further study. When the carbon source was glucose, the nitrogen source was ammonium chloride, the pH was 5, and the available phosphorus content was the highest. By exploring the possible mechanism of phosphorus release by phosphorus-solubilizing fungi, it was found that strain L4 produces several organic acids, such as oxalic acid, lactic acid, acetic acid, succinic acid, tartaric acid, malic acid, and citric acid. At 24 h, the alkaline phosphatase and acid phosphatase activities reached 154.72 mol/(L·h) and 120.99 mol/(L·h), respectively.

Plant Growth Promotion and Biocontrol Properties of Aneurinibacillus migulanus Isolated from Maize Roots (Zea mays)

Aims: To isolate Plant Growth Promoting Bacillus strain from maize roots, to evaluate its biocontrol potentials and to characterize the isolate using16S rRNA sequencing.&#x0D; Place and Duration of Study: Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University, Awka, between February 2019 and March 2020.&#x0D; Methodology: The isolation of Plant Growth Promoting Rhizobacteria (PGPR) from maize roots was done using Pikovskaya (PVK) agar. Quantitative determination of phosphate was carried out using PVK broth. Evaluations of other plant growth promoting properties were carried out such as IAA, etc. Fusarium and Enterobacter plant pathogens were isolated from diseased maize plants. The in vitro antagonism effects of the PGPR isolates against the pathogens were analyzed using the dual culture plate technique. The pot experiment was carried out in a completely randomized design. Plant characteristics such as plant height, shoot and root weight, chlorophyll content, as well as disease assessment were recorded accordingly. The organisms were identified using phenotypic and molecular methods.&#x0D; Results: Seven PGPR bacteria were isolated from maize (Zea mays) roots using PVK agar. Aneurinibacillus migulanus gave the highest solubilization index of 4.21 while isolate IS48 gave the lowest solubilization index of 1.47. A. migulanus produced IAA, ammonia and cellulase enzyme but no hydrogen cyanide. The organism showed antagonism activity against the two tested phytopathogens. In the pot experiment, A. migulanus treated plants showed a statistically insignificant difference in maize plant height at P=0.05 but gave significant increases in shoot and root wet weights. The organism offered 83.33% and 71.43% protection against Enterobacter and Fusarium pathogens respectively in the pot experiment.&#x0D; Conclusion: A. migulanus solubilized phosphate in addition to other plant growth promoting properties. It showed biocontrol potentials both in vitro and in vivo and thus can be used as substitute for synthetic agrochemicals.

ISOLATION AND CHARACTERIZATION OF ENDOPHYTIC ACTINOMYCETES ASSOCIATED WITH THYMUS SATUROIDES AND LAVANDULA MULTIFIDA GROWN IN BENI-MELLAL REGION IN MOROCCO

ABSTRACT The present study aims to isolate endophytic actinomycetes (EPM) that are able to tolerate abiotic stresses, solubilize tricalcium phosphate (TCP) and produce indole-3-acetic acid (IAA), amylase and cellulose activities, while exhibiting high antagonistic activity against Fusarium oxysporum. f. sp. albedinis. Endophytic actinomycetes were isolated from roots, stems and leaves of Thymus saturoides and Lavandula multifida plant grown in Beni Mellal region, Morocco. According to the macro- and micro-morphological characteristics, a total of eleven EPM isolates were isolated and purified. Results indicated that the majority of isolates were able to tolerate pH and salt stresses. Moreover, the majority of the isolates were able to solubilize TCP on Pikovskaya (PVK)-agar medium which ranged from 25% to 458%. Additionally, on PVK-liquid medium, soluble phosphate content varied from 163.81 µg/mL recorded for the isolate LRP1 to 730 µg/mL observed for the isolate TSRP2. For IAA production, the amount produced varied from 1.70 µg/mL recorded for the isolate LRP1 to 14.13 µg/mL recorded for both LTP2’ and TSRP2. Endophytic actinomycetes isolated in this work also showed a remarkable capacity to degrade some compounds such as cellulose and starch. In addition, some of them showed a strong inhibitory activity against the fungus F. albedinis with a percentage of inhibition ranged from 69.13% observed for LTP2’ to 86.6% recorded for the isolate LRP2. Results from this study indicate that the two isolates LTP2’ and TSRP2 were the most efficient and that actinomycetes could be used in biocontrol against the fungus F. albedinis.

Identification, characterization and optimization of phosphate solubilizing rhizobacteria (PSRB) from rice rhizosphere

Two billion people worldwide take rice (Oryza sativa L.) as a staple food. Phosphorus (P) and Nitrogen (N) are the major requirements of rice; although these are available in limited concentrations within rice growing regions. Among different types of Plant growth-promoting rhizobacteria (PGPR), Phosphate solubilizing rhizobacteria (PSRB) constitute an important class. These are known for plant growth promotion by enhancing P and N uptake. PSRB are nowadays used as biofertilizers to restore the soil health. Under the present investigation identification, characterization and optimization of phosphate solubilizing activity of these microbes at different pH, temperature and salt concentrations was carried out. Thirty-seven isolates were recovered from different regions of rice rhizosphere on Pikovskaya(PVK) agar among which 15 isolates were recovered from R.S. Pura, 12 isolates from Bishnah and 10 isolates were recovered from Akhnoor sector of Jammu, India. A prominent halo zone of clearance was developed around the colonies of 12 different isolates, indicating phosphate solubilization activity. Four distinct isolates were amplified, cloned and sequenced for taxonomic identification using 16S primers. The results indicated that PS 1, PS 2, PS 3, PS 4 were related to Pseudomonas aeruginosa, Bacillus subtilis strain 1, B. subtilis strain 2, B. subtilis strain 3, respectively. These strains when grown at a wide range of ecological factors showed maximum growth at pH between 6.8 and 8.8, temperature between 28 °C and 37 °C and salinity between 1% and 2%. Screening for phosphate solubilization activity revealed that the halo zone diameter formed by these isolates extended from 2.1 to 3.2 mm. The phosphate solubilizing efficiency (SE) ranged from 35.4 to 50.9 with highest value of 50.9 by PS4 and maximum P solubilization of 10.22 µg/ml was recorded by PS4 at 7th day. Phosphate solubilization activity of these identified PSRB strains can be utilized and explored in the rice growing belts of Jammu region which are deficient in phosphorus. MIC value for zinc sulphate heptahydrate in 12 isolates varied from 1 mg/ml to 6 mg/ml. Phosphate solubilization activity and MIC of these identified PSRB strains can be utilized and explored in the rice growing belts of Jammu region which are deficient in phosphorus.

Bacterial strains found in the soils of a municipal solid waste dumping site facilitated phosphate solubilization along with cadmium remediation

Phosphate solubilizing bacteria (PSB) that can withstand high cadmium (Cd) stress is a desired combination for bioremediation. This study evaluated the Cd bioremediation potential of four PSB strains isolated from the contaminated soils of a municipal solid waste (MSW) discarding site (Guwahati, India). PSB strains were cultured in Pikovskaya (PVK) media, which led to higher acid phosphatase (ACP) activity and the release of organic acid. Optical density (OD) measurements were performed to determine the growth pattern of PSB; furthermore, Cd uptake by PSB was evaluated using infrared spectroscopy (IR) and X-Ray Diffraction (XRD) analyses. The 16S rRNA taxonomic analysis revealed that all the four promising PSB strains belonged to either Bacillus sp. or Enterobacter sp. One strain (SM_SS8) demonstrated higher tolerance towards Cd (up to 100 mg L−1). Flow cytometry analysis revealed 70.92%, 46.93% and 20.4% viability of SM_SS8 in 10, 50 and 100 mg L−1, respectively in PVK media containing Cd. This study has therefore substantiated the bioremediation of Cd from polluted soil by the PSB isolates. Thus, experimental results revealed a potential combo benefit, phosphate solubilization along with Cd remediation.

Characterization and Plant Growth Promoting Properties of a Bacillus sp. Isolated from Maize Roots

Aim: To isolate plant growth promoting Bacillus strains from maize roots and to characterize using molecular methods, the strain with greatest potential for plant growth promotion.&#x0D; Place and Duration of Study: Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University, Awka, between February 2019 and March 2020.&#x0D; Methodology: The isolation of plant growth promoting rhizobacteria (PGPR) from maize roots was made using Nitrogen Free Bromothymol Blue (NFB) broth. They were screened for Phosphate solubilizing activities on Pikovskaya (PVK) agar. Quantitative determination and solubilization of different types of Phosphates was carried out using Pikovskaya broth. Optimization of factors affecting phosphate such as NaCl concentration, initial pH of the medium, size of inoculum, was done using pvk broth. Evaluation of other plant growth promoting properties were carried out such as IAA, Ammonia, cellulase and HCN production. &#x0D; Results: Eleven Nitrogen fixing bacteria were isolated using NFB broth based on colour change of the medium from green to blue. Test for phosphate solubilization abilities of the organisms revealed that nine of the isolates solubilized phosphate on PVK agar. Organism coded with IS52 gave the least solubilization index of 1.14 while isolate IS19 gave the highest index of 3.4. Isolate IS19 yielded the highest amount of 73.5µg/ml P, while isolate IS30 was the weakest solubilizer in PVK broth, yielding 19.4µg/ml P. The best isolate IS19, produced the plant growth hormone Indole Acetic acid at a concentration of 105.4μg/ml. The organism also gave a positive result for ammonia and cellulase production but did not produce Hydrogen cyanide. It was identified as Bacillus subtilis using the 16S rRNA gene sequencing.&#x0D; Conclusion: Bacillus subtilis fixed Nitrogen qualitatively and solubilized insoluble phosphates in addition to other plant growth promoting properties, thus Bacillus subtilis has potential for plant growth promotion, making it an efficient strain for biofertilizer production

Screening and optimization of indole-3-acetic acid production and phosphate solubilization by rhizobacterial strains isolated from Acacia cyanophylla root nodules and their effects on its plant growth

BackgroundPlant growth-promoting rhizobacteria (PGPR) are known to improve plant growth and are used as biofertilizers, thanks to their numerous benefits to agriculture such as phosphorus solubilization and phytohormone production. In this paper, four rhizospheric bacteria (Phyllobacterium sp., Bacillus sp., Agrobacterium sp., and Rhizobium sp.) isolated from surface-sterilized root nodules of Acacia cyanophylla were tested for their ability to solubilize inorganic phosphate and to produce indole-3-acetic acid (IAA) under laboratory conditions. Then, the best IAA producer (Rhizobium sp.) was selected to test optimized conditions for IAA production. Finally, the effect of the four strains on plant growth for A. cyanophylla was evaluated in vivo. ResultsThe results showed that the totality of the tested isolates had solubilized inorganic phosphate (P) in both NBRIP (National Botanical Research Institute Phosphate) and PVK (Pikovskaya) media. Bacillus sp. was a high P-solubilizer and showed maximum solubilization in PVK (519 μg ml-1) and NBRIP (782 μg ml-1). The optimization of maximum phosphate solubilization was done using different sources of carbon (1%) and nitrogen (0.1%). Glucose and ammonium sulfate were selected to be the best carbon and nitrogen source for phosphate solubilization by all tested strains, except for Phyllobacterium sp., which recorded the highest phosphate solubilization with ammonium nitrate. The IAA production by the tested strains indicated that Rhizobium sp. produced the highest amount of IAA (90.21 μg ml-1) in culture media supplemented with L-tryptophan. The best production was observed with L-Trp concentration of 0.2% (116.42 μg ml-1) and at an initial pH of 9 (116.07 μg ml-1). The effect of NaCl on IAA production was tested at concentrations of 0 to 5% and the maximum production of 89.43 μg ml-1 was found at 2% NaCl. The extraction of crude IAA from this strain was done and purity was confirmed with Thin Layer Chromatography (TLC) analysis. A specific spot from the extracted IAA production was found to correspond with a standard spot of IAA with the same Rf value. Finally, the tested PGPR demonstrated growth stimulatory effects on Acacia cyanophylla seedlings in vivo, with a great increase of shoots’ and roots’ dry weights, and shoot length compared to control.The rhizobacterial isolates were identified by 16S rDNA sequence analysis as Agrobacterium sp. NA11001, Phyllobacterium sp. C65, Bacillus sp. CS14, and Rhizobium sp. V3E1. ConclusionThis study highlights the importance of the use of phosphate solubilizing and IAA producer microorganisms as biofertilizers to increase crop yields. The studied strains showed a significant phosphate solubilization potential and IAA production. The use of selected strains as inoculants would be interesting, in particular with a view of promoting sustainable agriculture. However, further studies to verify the efficacy of the best isolates in situ is certainly required.

Alcaligenes aquatilis GTE53: Phosphate solubilising and bioremediation bacterium isolated from new biotope “phosphate sludge enriched-compost”

The isolation and identification of beneficial bacteria from the active phase of composting is considered to be a key bio-quality parameter for the assessment of the process. The aim of this work was the selection and identification of beneficial bacteria from a co-composting experiment of vegetable waste (VW), olive oil mill waste (O2MW), and phosphate sludge (PS). Phosphate-solubilizing strains were isolated from the thermophilic phase using Pikovskaya (PVK) solid medium supplemented with tricalcium phosphate Ca3(PO4) (TCP) as the sole source of phosphorus (P). Therefore, the selected isolate Alcaligenes aquatilis GTE53 was tested to tolerate abiotic stresses (different levels of temperature, variable pH, high salinity and water stress). The isolate was also assessed for indole acetic acid (IAA) and siderophores synthesis, nitrogen fixation, phenol degradation and pathogens inactivation. The quality of the co-composting process was also investigated by monitoring the physico-chemical parameters. The obtained results showed that A. aquatilis GTE53 displayed a higher solubilization index of 2.4 and was efficiently dissolved, up to 162.8 and 247.4 mg·mL−1 of inorganic phosphate from PS and phosphate rock (PR), respectively. A. aquatilis GTE53 exhibited siderophores and IAA release, along with atmospheric nitrogen fixation. In addition to that, A. aquatilis GTE53 showed a high resistance to heat and tolerance to acidic and alkaline pH, high salinity and water stress. Moreover, A. aquatilis GTE53 could degrade 99.2% of phenol from a high-concentrated medium (1100 mg·L−1 of phenol) and can inactivate the most abundant pathogens in industrial wastes: Escherichia coli, Streptococcus sp., Salmonella sp., and Fusarium oxysporum albedinis. Analysis of temperature, pH, electrical conductivity, carbon/nitrogen (C/N) ratio, indicated successful co-composting. An efficient transformation of P to the available form and a great abatement of polyphenols, were also recorded during the process. The findings of this study will help to advance the understanding of A. aquatilis GTE53 functions and will facilitate its application to promote beneficial microbial organisms during composting, thus obtaining a high-quality product.

Screening of Phosphate-Solubilizing Fungi From Air and Soil in Yunnan, China: Four Novel Species in Aspergillus, Gongronella, Penicillium, and Talaromyces.

Phosphate-solubilizing fungi (PSF) play an important role in increasing the bioavailability of phosphorus in soils for plants. Thirteen fungal strains, one collected from air and 12 from soil, were screened and described here in detail. These fungal strains were tested for their ability to solubilize tricalcium phosphate (TCP) on both solid and liquid Pikovskaya (PVK) media in vitro. The airborne fungal strain KUMCC 18-0196 (Aspergillus hydei sp. nov.) showed the most significant phosphate solubilizing activity on a solid PVK medium with the solubilization index (SI) (2.58 ± 0.04 cm) and the highest solubilized phosphates (1523.33 ± 47.87 μg/mL) on a liquid PVK medium. To the best of our knowledge, A. hydei sp. nov. is the first phosphate-solubilizing fungus reported from air. We also provide the identification especially for Aspergillus, Penicillium and Talaromyces, generally reported as PSF. It is important to not only screen for PSF but also identify species properly so that researchers have a clearer taxonomic picture for identifying potential taxa for future plant growth-promoting applications. Herein, A. hydei (section Nigri), Gongronella hydei, Penicillium soli (section Lanata-Divaricata) and Talaromyces yunnanensis (section Talaromyces) are fully described and introduced as new to science. These four new species are identified based on both morphological characteristics and multigene phylogenetic analyses, including the genealogical concordance phylogenetic species recognition method where necessary. Penicillium austrosinense is considered to be a synonym of P. guaibinense.

Biosolubilization of Low-Grade Rock Phosphate by Native Microbial Consortia from Phosphate Mines: Effect of Sampling Sources and Culture Media

Two native microbial consortia were isolated from the soil and drainage in the phosphate mines, and their abilities to solubilize low-grade rock phosphate (RP) in two different culture media, namely Pikovskaya (PKV) and National Botanical Research Institute’s phosphate (NBRIP) medium, respectively, were estimated. Results showed that the two microbial consortia could grow steadily in the solution and continuously released soluble phosphate from the RP during 14 d of experiments. This process was accompanied by a drop in pH of the solution. The microbial consortium isolated from the soil achieved the largest release of soluble phosphate and pH reduction in the PKV medium among the four microbial consortia named PS, NS, PD, and ND, respectively. Fourier transform infrared spectroscopy (FTIR) analysis of RP indicated that hydroxyl, alkyl C-H bond, amide, and carboxyl were involved in the RP solubilization, and significant changes of them were observed after the experiments. The four microbial consortia were sampled before and after the experiments for the analysis of their bacterial and fungal community structures by Illumina MiSeq sequencing. Results showed that the relative diversities and abundances of the dominant bacteria and fungi varied with different sampling sources (soil and drainage) and culture media (PKV and NBRIP medium).

Characteristics of Inorganic Phosphate-Solubilizing Bacteria from the Sediments of a Eutrophic Lake.

Inorganic phosphate-solubilizing bacteria (IPB) are an important component of microbial populations in lake sediments. The phosphate that they decompose and release becomes an important source of phosphorus for eutrophic algae. The IPB strains were screened and isolated from the sediments of Sancha Lake using National Botanical Research Institute’s phosphate (NBRIP) plates. Their taxonomy was further determined by the 16S rDNA technique. The tricalcium phosphate-solubilizing ability of obtained IPB strains was evaluated using NBRIP- bromophenol blue (BPB) plates and Pikovskaya (PVK) liquid medium. Then, the ability of IPB strains to release phosphorus from the sediments were investigated by mimicking the lake environment. In this study, a total of 43 IPB strains were screened and isolated from the sediments of Sancha Lake, belonging to three phyla, eight families, and ten genera. Among them, two potentially new strains, SWSI1728 and SWSI1734, belonged to genus Bacillus, and a potentially new strain, SWSI1719, belonged to family Micromonosporaceae. Overall, the IBP strains were highly diverse and Bacillus and Paenibacillus were the dominant genera. In the tricalcium phosphate-solubilizing experiment, only 30 of the 43 IPB strains exhibited clear halo zones on plates, while in the liquid culture experiment, all strains were able to dissolve tricalcium phosphate. The phosphate-solubilizing abilities of the strains varied significantly, and the strain SWSI1725 of the Bacillus genus showed the strongest ability with a phosphate-solubilizing content of 103.57 mg/L. The sterilized systems demonstrated significantly elevated phosphorus hydrochloride (HCl–P) decomposition and release from the sediments after the inoculation of IPB strains, whereas no significant effect was demonstrated on the phosphonium hydroxide (NaOH-P). Thus, the IPB strains in the sediments of Sancha Lake possessed rich diversity and the ability to release phosphorus in sediments.

Solubilization of inorganic phosphate by rhizospheric fungi isolated from soil cultivated with Sorghum bicolor L.

Three zinc-phosphate-solubilizing fungi (ZPSF) were isolated from rhizospheric soil cultivated with Sorghum bicolor L. The fungal isolates were identified as Aspergillus chevalieri, Fusarium moniliforme, and Trichoderma harzianum. The results showed that halo zone formation by ZPSF on Pikovskaya (PVK) agar medium plates was an indicator of zinc phosphate (ZP) solubilization. The lowest in pH of the inoculated medium containing ZP was observed with A. chevalieri, followed by T. harzianum, followed by F. moniliforme, compared with the initial pH (6.5) of the non-inoculated medium. ZP solubilization processes at different temperatures (10 °C, 20 °C, 30 °C, and 40 °C) were conducted using ZPSF at different doses of ZP (0.5 g/L, 1 g/L, and 2 g/L). The released P, to P2O5, was monitored during the solubilization process. The released phosphorus increased as the temperature increased, with the greatest values of phosphorus obtained with F. moniliforme, A. chevalieri, and T. harzianum being 11.54 mg/L, 24.40 mg/L, and 28.40 mg/L, at 30 °C and a dose of 2 g/L of ZP, respectively. In contrast, the smallest values of phosphorus were 11.89 mg/L, 8.2 mg/L, and 7.97 mg/L, at 10 °C and a dose of 0.5 g/L of ZP, with F. moniliforme, A. chevalieri, and T. harzianum, respectively.

Screening for Lotus creticus growth promoting rhizobacteria under greenhouse conditions

Utilization of plant growth promoting rhizobacteria (PGPR) is now gradually increasing in agriculture and offers an attractive way to replace chemical fertilizers, pesticides, and supplements. This study was conducted with a view to isolate bacteria from the rhizosphere of the legume Lotus creticus ( L. creticus ) and to assess their plant growth promoting functional potentialities. A total of 113 rhizobacteria was isolated from the rhizosphere of L. creticus and were tested for their capacity of solubilizing tricalcium phosphate (TCP) on Pikovskaya (PVK) solid medium. Out of 29 phosphate solubilizing bacteria (PSB), 5 isolates were selected for their solubilization diameters (between 0.6 and 1.5 cm). These isolates were characterized for plant growth promoting (PGP) traits. The results showed that the highest concentration of indole acetic acid (IAA) was produced by LCR33 (19.08 ± 0.96 mg L -1 ). All 5 isolates could produce hydrogen cyanide (HCN), siderophores, ammonia and amino-cyclopropane carboxylate (ACC) deaminase. The isolates were evaluated for TCP solubilizing quantitative assay in PVK liquid medium. The concentrations of solubilized P were between 43.34±0.18 mg L -1 and 173.57±0.77 mg L -1 . This solubilization was accompanied by a pH decrease of the culture media from 7 to 4.06. Furthermore, the 5 selected PSB were tested in vitro for antagonism against phytopathogenic fungus Fusarium oxysporum . In fact, all the PSB, were capable of inhibiting its growth and the highest percentages of inhibition were obtained for LCP27 and LCR33 (48.15±0.99% and 40.74±0.45%). Also, the effect of these 2 PSB on growth of L. creticus plants was investigated under greenhouse conditions. Significant increases were obtained for shoot and root length and dry and fresh matter production of plants as compared to the uninoculated control. These PSB could be recommended as biofertilizers for contributing to the rehabilitation of degraded soils.

Soil extract calcium phosphate media for screening of phosphate-solubilizing bacteria

Media development for screening bacteria capable of solubilizing inorganic complex forms of phosphorous to a soluble form is a major concern in agricultural research. This study formulated an effective, alternative solid and liquid media to the currently available Pikovskaya (PVK) medium, which relies on the use of calcium phosphate and numerous inorganic components for the screening of phosphate-solubilizing bacteria. In this present study, soil extract calcium phosphate (SECP) broth was formulated with simple medium requirements. SECP broth performed better than PVK broth as evidenced by its ability to support a higher bacterial population, higher reduction in broth pH and higher organic acid production and ultimately a better phosphate solubilization, based on the zone of clearance using bromocresol green or congo red in addition to bromothymol blue. Apart from calcium phosphate, rock phosphate and aluminum phosphate were also used as phosphate sources for the screening of different phosphate-solubilizing strains. This solid SECP medium could be used effectively with other phosphorous sources such as rock phosphate and aluminum phosphate. A high correlation between solid-based assay and broth-based assay for screening of phosphate-solubilizing bacteria was observed in the study.

Physiological characterization of rhizobial strains nodulating alfalfa (Medicago sativa) isolated from soils of Southeastern Morocco

The legume-rhizobia symbiosis provides the necessary nitrogen for plants growth and contributes to the improvement of soil nitrogen level. However, environmental constraints prevailing in many Moroccan regions constitute the limiting factors for this symbiotic interaction. For this reason, the present work aims to select some rhizobial strains nodulating alfalfa ( Medicago sativa L.) for their performance under environmental stresses. Rhizobial strains nodulating alfalfa were isolated from soils of Tafilalet region ( Arfoud 1 , Arfoud 4 , Aoufous and Rich ) and purified in yeast extract mannitol (YMA) medium. After nodulation test, 20 strains were able to induce root nodulation in alfalfa and they were tested for their tolerance to high salinity, water deficit, acidity and temperature. These strains were also assessed for their abilities to solubilize inorganic phosphorus using Pikovskaya (PVK) medium. The results indicated that the studied strains developed different behaviors in response to all considered abiotic stresses. The positive correlations between strains behaviors and origin were noted. In general, strains from Arfoud1 and Arfoud 4 were qualified as the most tolerant to salt stress and can grow under extreme pH levels (4 to 10). Whereas, strains from Rich were the most tolerant to water deficit. Results recorded on PVK medium mentioned that some strains ( Arf1RL1 , RYRL9 and Arf4RL16 ) showed a high potential of inorganic phosphorus solubilization. Some strains such as Arf1RL1 and Arf4RL13 both high tolerance level to considered environmental constraints and high potential of inorganic phosphorus solubilization.

Phosphate-solubilizing fungi for efficient soil phosphorus management

Rhizosphere soil extracts obtained from eight cinnamon species and three pepper species cultivated organically were inoculated initially on Pikovskaya (PVK) medium. The twelve fungal isolates that solubilized the insoluble phosphates were further tested on the same medium added with both insoluble tricalcium phosphate and soluble dipotassium hydrogen phosphate to detect the expression of the trait in the presence of soluble phosphates in soil, for which all twelve qualified. The efficiencies of phosphate solubilization were investigated in two liquid media: PVK medium to represent soil phosphate pool and a rock phosphate (RP) medium, by determining the available phosphorus concentration in the liquid media at the end of the 24 h and 72 h incubation periods, respectively. The pH reduction in the medium was measured to ascertain the organic acid production by fungal isolates as a mechanism of solubilization. Three fungal isolates showing the highest efficiencies in phosphate solubilization were taxonomically identified based on the micromorphological characteristics and molecular techniques. Two isolates were identified as Penicillium oxalicum and Trichorderma virens and the other was belonging to the genus Aspergillus. A possible synergism between the three fungal species towards phosphate dissolution was detected using the broth culture procedure. Aspergillus sp. in combination with P. oxalicum dissolved both Ca3(PO4)2 and RP recording the highest significant dissolved phosphate levels of 893.43 (±56.768) mg P/L and 309.42 (±42.52) mg P/L, respectively, within 72 h post inoculation, making them prospective candidates for increased phosphate availability of soil phosphate pool and rock phosphates.

The Potential Use of Plant Growth-Promoting Rhizobacteria (PGPR) as Antagonists and Biocontrol Agents in Hevea Diseases

The rhizosphere related bacterial species have a significant capability in plant growth promotion, referred to as Plant Growth Promoting Rhizobacteria (PGPR). Several substances produced by antagonistic rhizobacteria have been related to pathogen control and indirect promotion of growth in many plants. The objectives of this study were to identify effective PGPR bacteria in rubber soils of Sri Lanka, determine the potential use as antagonists and biocontrol agents to control economically important Hevea diseases in nurseries, and identify effective novel inoculant formulations. Soil samples were collected from the root rhizozphere of cover crops in Hevea fields in Rubber Research Institute of Sri Lanka (RRISL), Dartonfield. Bacteria were isolated by TSA plate method. Isolated PGPR bacteria were subjected to test the enzyme secretion ability. Pectinase, Lipase, and Amylase secretion ability of PGPR were tested in specifically developed media. Auxin production of PGPR were measured using freshly prepared Salkowsky reagent. Phosphorous solubilizing ability of PGPR was tested using Pikovskaya (PVK) medium. Antagonistic ability of PGPR was tested against the Rigidoporous microporus, Fusarrium sp. and Pythium sp. causing white root disease and Patch Canker disease in rubber respectively. Paper disc diffusion method was used to test the antagonism of selected bacteria and the percentage growth inhibition was calculated. Plasmid DNA extraction of antagonistic PGPR was done following the alkaline SDS method. Gene sequencing of selected bacteria was further carried out. Among 13 selected PGPR bacterial species, 3 bacteria were showing significant antagonism towards R. microporus. (Percentage growth inhibition by 51.2%, 46.8% and 44.3% respectively) Fusarium sp. was controlled by 7 PGPR species. Pythium sp. was controlled by 2 PGPR (Percentage growth inhibition by 41.6% and 38.7% respectively) 5 types of combinations of bacteria were tested. Among 5 types, 2 types were highly effective in control of R. microporus (Percentage growth inhibition by 79.8% and 72.6% respectively). All 5 combinations were moderately effective in control Fusarium sp. and 1 type was highly effective in control of Pythium sp. (Percentage growth inhibition by 67.8%). PGPR can be used as effective biocontrol agents in control of Hevea pathogens. The combinations of PGPR are more effective than the individual bacterial species. This will be leading to improve the productivity of Hevea plantations in Sri Lanka. Keywords: Plant Growth Promoting Rhizobacteria, Antagonism, Hevea pathogens

Trichoderma virens Mediated Rock Phosphate Solubilization: A Potential Replacement For Triple Super Phosphate in Rice Cultivation

Annually, Sri Lanka spends 20 billion Rupees to import Triple super Phosphate (TSP) mainly for the rice cultivation. It has been recorded that, significantly high arsenic content is present in imported TSP compared to Eppawala Rock phosphate (ERP). Further, the current understanding is that arsenic is among the major etiological agents of Chronic Kidney Disease. Hence, a progressive reduction in the use of TSP in rice cultivation may help in reducing health and environmental problems associated with it. Yet the major constraint in the use of ERP in rice cultivation is its low solubility. As microorganisms are efficient solubilizers of Rock phosphate, attempts were made to employ a local isolate of Trichodrema virens, which has a proven track record for ERP solubilization, to increase the solubility of ERP with the aim of developing an environmentally friendly liquid P fertilizer. A pot experiment was conducted at Rice Research and Development Institute at Batalagoda. An amended Pikovskaya (PVK) medium with ERP was inoculated with T. virens containing 1X 106 spores/ml and incubated at 100 rpm oscillation until maximum solubilized P content was obtained. The filtered and autoclaved culture filtrate was applied to the rice variety Bg 250 one week before transplanting. Fertilizer application included: Control without any fertilizer (C1), control with recommended level of MOP, urea and no P (C2), Control with PVK medium without P source (C3), TSP at recommended level (P1), TSP at half recommended level together with developed fertilizer added to replace half recommended level of TSP quantitatively (P2), TSP at half recommended level (P3), developed fertilizer applied at recommended level of TSP (P4). Additionally, the recommended level of MOP and Urea was applied to every treatment except for C1. Completely randomized design was employed with 12 replicates and each pot containing 4 rice plants. Plant growth parameters and plant and soil P contents were quantitatively determined by destructive sampling of 04 replicates from each treatment at tillering stage and at 50% flowering stage while yield parameters with plant and soil P were measured at harvesting stage. The results obtained were analyzed using T test and one way ANOVA. During fertilizer development, maximum P content (42.55 mg P/l) was obtained at 72 hour post incubation. All the studied parameters differed significantly among treatments (P = 0.000). Superior growth and yield performance were shown by P2 where the performance was not significantly different from that of P1 for all the tested parameters. Even though treatment P4 was not superior to treatment P1, it was significantly superior to P3. Hence, the data suggests the possibility of reducing the recommended level of TSP into half by using the developed liquid fertilizer to minimize TSP usage. Keywords: Trichodrema virens, Phosphorus, Eppawala rock phosphate, Rice, Growth parameters, Yield parameters