Isolation and characterisation of phosphate-solubilising bacteria from serpentine soils in Yen Bai Province

Phosphorus is the least mobile element in plant and soil contrary to other macronutrients. Phosphorus solubilizing bacteria play role in phosphorus nutrition by enhancing its availability to plants. Aim of study was to deal with isolation, characterization and identification of phosphate solubilizing bacteria (PSBs) from 15 paddy rice soil samples collected in rice–shrimp rotational farming system in salt affected soil areas of the Mekong Delta of Vietnam including Bac Lieu, Ca Mau, Soc Trang, Kien Giang and Ben Tre by using NBRIP media containing 1% NaCl. Concentration of phosphate was determined by method of colometric determination of molybdate. Results showed that 95 isolates indicating potentially as PSBs through a exhibiting of a halo zone happening around bacterial colonies on NBRIP media. Twenty out of ninety five isolates showed their highly phosphate solubilizing ability (>1000 mg.L-1) in the liquid culture. Bacterial strain coded as BL1-10 solubilized maximumly 2044 mg.L-1 phosphate in liquid media after 5 incubation days. The cell number and phosphate solubilizing ability of Bl-10 strain were till good and not affected under environmental coditions like 40oC, salinity range between 0.5 and 5 % NaC and pH range between 3 and 5. Three kinds of trade name fungicides like DA roral, Topsin M và Antracol 70WP did not affect on cell numbers and phosphate solubilizing ability of Bl-10 strain. However, cell numbers and function of phosphate solubilizing ability of this bacterial strain was completely inhibited by Ridomil Gold fungicide. This strain was genetically identified as species of Burkholderia sp. BL1-10 based on results of the 16S rRNA gene sequence analysis. In conclusion, the study suggests the most promising phosphate solubilizing bacteria, Burkholderia sp. BL1-10 can be used as efficient biofertilizer inoculant to promote plant growth.

Phosphorus availability is a major limiting for crop production. Bacterial solubilization of insoluble inorganic phosphate has been studied as a means of providing available phosphorus for crop production. Bacterial abilities to solubilize calcium phosphate and rock phosphate have been identified to be related with their abilities to produce gluconic acid and ketogluconic acid. However, there is no information regarding the relationship between bacterial ability to solubilize aluminum phosphate and their ability to produce organic acids. This study was conducted to investigate the relationship between bacterial ability to solubilize calcium and aluminum phosphates with their ability to produce organic acids. Bacterial ability to solubilize calcium and aluminum phosphates were determined as the concentration of soluble phosphate in the filtrate of bacterial cultivation media, while bacterial ability to produce organic acids were assessed from the accumulated organic acids in its. The results showed that bacterial abilities to solubilize calcium and aluminum phosphates well related to their abilities to produce organic acids. Organic acids related with the solubilization of calcium phosphate differ from the ones relatedAlam, S., S. Khalil, N. Ayub, and M. Rashid. 2002. In vitro solubilization of inorganic phosphate by phosphate solubilizing microorganisms (PSM) from maize rhizosphere. Int. J. Agri. Biol. 4: 454-458.Beauchemin, S., D. Hesterberg, J. Chou, M. Beauchemin, R.R. Simard, and D.E. Sayers. 2003. Speciation of phosphorus in phosphorus-enriched agricultural soils using X-ray absorption near-edge structure spectroscopy and chemical fractionation. J. Environ. Qual. 32:1809–1819.Bolan, N.S., R. Naidu, S. Mahimairaja, dan S. Baskaran. 1994. Influence of low-molecular-weight organic acids on the solubilization of phosphates. Biol. Fertil. Soils 18: 311-319.Cline, G.R., P.E. Powell, P.J. Szaniszlo, dan C.P. Reid. 1983. Comparison of the abilities of hydroxamic and other natural organic acids to chelate iron and other ions in soil. Soil Sci. 136: 145-157.Curtin, D., and J.K. Syers, 2001. Lime-induced changes in indices of soil phosphate availability. Soil Sci. Soc. Am. J. 65:147–152.Fox, T.R., N.B. Comerford, dan W.W. McFee. 1990. Phosphorus and aluminium release from a spodic Horizon mediated by organic acids. Soil Sci. Soc. Am. J. 54: 1763-1767.Hue, N.V., G.R. Craddock, dan F. Adams. 1986. Effect of organic acids on aluminium toxicity in subsoils. Soil Sci. Soc. Am. J. 50: 28-34.Johnson, S.E., and R.H. Loeppert. 2006. Role of organic acids in phosphate mobilization from iron oxide. Soil Sci. Soc. Am. J. 70:222–234.Kumari, A., K.K. Kapoor, B.S. Kundu, and R.K. Mehta. 2008. Identification of organic acids produced during rice straw decomposition and their role in rock phosphate solubilization. Plant Soil Environ. 54: 72–77Lopez-Hernandez, D., D. Flores, G. Siegert, dan J.V. Rodriguez. 1979. The effect of some organic anions on phosphate removal from acid and calcareous soils. Soil Sci. 128: 321-326.Lopez-Pineiro, A., dan A. Garcia-Navarro. 2001. Phosphate fractions and availability in Vertisols of South-Western Spain. Soil Sci. 166: 548-556.Olsen, S.R. dan Sommers, L.E. 1982. Phosphorus. In Page, A.L., Miller, R.H. & Keeney, D.R. (eds.). Methods of Soil Analysis. Part 2. 2nd ed. ASA and SSSA Publisher, Madison.Rao, W.V.B.S., and M.K. Sinha. 1963. Phosphate dissolving microorganisms in the soil and rhizosphere. Indian J. agric. Sci. 33: 272-278.Rodriguez, H., T. Gonzalez, I. Goire, dan Y. Bashan. 2004. Gluconic acid production and phosphate solubilization by the plant growth-promoting bacterium Azospirillum spp. Naturwissenschaften 91: 552-555.Sagoe, C.I., T. Ando, K. Kouno, and T. Nagaoka. 1997. Effect of organic-acid treatment of phosphate rocks on the phosphorus availability to Italian ryegrass. Soil Sci. Plant Nutr. 43: 1067-1072.Sanchez, P.A. 1976. Properties and Management of Soils in the Tropics. John Wiley & Sons, Inc. New York. 618 pp.Siddique, M.T., and J.S. Robinson. 2003. Phosphorus sorption and availability in soils amended with animal manures and sewage sludge. J. Environ. Qual. 32:1114–1121.Song, O.R., S.J. Lee, Y.S. Lee, S.C. Lee, K.K. Kim, dan Y.L. Choi. 2008. Solubilization of insoluble inorganic phosphate by Burkholderia cepacia DA23 isolated from cultivated soil. Braz. J. Microbiol. 39: 151-156.Sridevi, M., K.V. Mallaiah, and N.C.S. Yadav. 2007. Phosphate solubilization by Rhizobium isolates from Crotalaria species. J. Plant Sci. 2: 635-639.Traina, S.J., G. Sposito, D. Hesterberg, dan U. Kafkafi. 1986. Effects of pH and organic acids on orthophosphate solubility in an acidic, montmorillonitic soil. Soil Sci. Am. J. 50: 45-52.Trivedi, P., and T. Sa. 2008. Pseudomonas corrugata (NRRL B-30409) mutants increased phosphate solubilization, organic acid production, and plant growth at lower temperatures. Curr. Microbiol. 56: 140-144.Tunesi, S., V. Poggi, and C. Gessa. 1999. Phosphate adsorption and precipitation in calcareous soils: The role of calcium ions in solution and carbonate minerals. Nutr. Cycling Agroecosyst. 53:219–227.Zhang, M., A.K. Alva, Y.C. Li, dan D.V. Calvert. 2001. Aluminium and iron fractions affecting phosphorus solubility and reactions in selected sandy soils. Soil Sci. 166: 940-948.with the solubilization of aluminum phosphate. Moreover, there is similarity in the production of organic acids related to the solubilization of aluminum phosphates and iron phosphate.

Laboratory-scale simulation study on the bioremediation of marine oil pollution by phosphate-solubilizing bacteria Bacillus subtilis PSB-1.

Soil microorganisms play an important role in maintaining natural ecological balance through active participation in carbon, nitrogen, sulfur, and phosphorous cycles. Phosphate-solubilizing bacteria (PSB) are of high importance in the rhizosphere, enhancing the solubilization of inorganic phosphorus complexes into soluble forms available for plant nutrition. The investigation of this species of bacteria is of major interest in agriculture, as they can be used as biofertilizers for crops. In the present study, 28 isolates of PSB were obtained after the phosphate enrichment of soil samples from five Tunisian regions. Five PSB species were identified by 16S rRNA gene sequencing including Pseudomonas fluorescens, P. putida, and P. taiwanensis, Stenotrophomonas maltophilia, and Pantoea agglomerans. Solid and liquid Pikovskaya’s (PVK) and National Botanical Research Institute’s (NBRIP) media containing insoluble tricalcium phosphate were used for the evaluation of the phosphate solubilization ability of the bacterial isolates by two methods: visual evaluation of the solubilization zone around colonies (halo) and determination of solubilized phosphates in liquid medium by the colorimetric method of the vanado-molybdate yellow. Based on the results of the halo method, the isolate of each species that showed the higher phosphate solubilization index was selected for evaluation of phosphate solubilization by the colorimetric method. In the liquid media, the bacterial isolates showed phosphate solubilization ranging from 535.70 to 618.57 µg mL−1 in the NBRIP medium, and 374.20 to 544.28 µg mL−1 in the PVK medium, with the highest values produced by P. fluorescens. The best phosphate solubilization ability and higher reduction in broth pH, which indicates higher organic acid production, were achieved in NBRIP broth for most of the PSB. Strong correlations were observed between the average capability of PSB to solubilize phosphates and both the pH and total phosphorous content in the soil. The production of the hormone indole acetic acid (IAA), which can promote plant growth, was observed for all five PSB species. Among them, P. fluorescens obtained from the forest soil of northern Tunisia showed the highest production of IAA (50.4 ± 0.9 µg mL−1).

Phosphate solubilizing and nitrogen-fixing bacteria have a vital role in improving soil fertility and reverting adversely affected soil properties. These bacteria could contribute towards sustainable agriculture with a focus on reducing excessive use of commercial fertilizers. This study aimed at investigating autochthonous populations of phosphate solubilizing and nitrogen-fixing bacteria from Lake Ol’Bolossat sediments. The total microbial counts ranged between 4.8 x 103 to 8.5 x 105 cfu/ml. A total of 50 bacteria were isolated, 34 were obtained from Pikovskaya’s agar medium while 16 were obtained from Norris Glucose Nitrogen free medium. Based on morphological and 16S rRNA gene analyses, the isolates were clustered under the genera Bacillus, Arthrobacter, Pseudomonas, Paenibacillus, Fictibacillus and Acinetobacter. Among potentially novel strains, four strains NFDA2, PKGBC1 (MT799539), PKGB5 and SCEC2 (MT799543) belonged to genus Bacillus, three strains NFGA1 (MT799529), NFGA4 and SCDB3 belonged to the genus Pseudomonas, two strains NFEB6 (MT799528) and NFDC5 belonged to the genus Paenibacillus, one strain PKHC3 (MT7995441) belonged to the genus Arthrobacter while one strain NFDC4, belonged to the genus Acinetobacter. Generally, the phosphate solubilizing bacteria were the most diverse and genera Bacillus, Fictibacillus and Pseudomonas were the most dominant, however, nitrogen-fixing bacteria were dominated by genera Arthrobacter and Pseudomonas.

Serpentine soils are characterized as a unique environment with low nutrient availability and high heavy metal concentrations, often hostile to many plant species. Even though these unfavorable conditions hinder the growth of various plants, particular vegetation with different adaptive mechanisms thrives undisturbed. One of the main contributors to serpentine adaptation represents serpentine bacteria with plant growth-promoting properties that assemble delicate interactions with serpentine plants. Robinia pseudoacacia L. is an invasive but adaptive species with phytoremediation potential and demonstrates extraordinary success in this environment. To explore more in-depth the role of plant growth-promoting serpentine bacteria, we isolated them and tested their various plant growth-promoting traits both from the rhizosphere and roots of R. pseudoacacia. Based on the demonstrated plant growth-promoting traits such as siderophore production, phosphate solubilization, nitrogen fixation, indole-3-acetic acid production, and ACC deaminase production, we sequenced overall 25 isolates, 14 from the rhizosphere and 11 from the roots. Although more efficient in exhibiting plant growthpromoting traits, rhizospheric bacteria showed a low rate of diversity in comparison to endophytic bacteria. The majority of the isolates from the rhizosphere belong to Pseudomonas, while isolates from the roots exhibited higher diversity with genera Pseudomonas, Bacillus, Staphylococcus, Lysinibacillus and Brevibacterium/Peribacillus/Bacillus. The capacity of the described bacteria to produce siderophores, solubilize phosphate, and fix nitrogen highlights their central role in enhancing nutrient availability and facilitating R. pseudoacacia adaptation to serpentine soils. The findings highlight the potential significance of serpentine bacteria, particularly Pseudomonas, in contributing to the resilience and growth of R. pseudoacacia in serpentine environments.

Phosphate solubilization and acid phosphatase activity of Serratia sp. isolated from mangrove soil of Mahanadi river delta, Odisha, India.

Nugraha et al, 2019. Solubilization of Inorganic Phospate by Burkholderia spp. Associated with Oil Palm Rhizosphere in Mineral Acid Soil. JSLO 8(1):86-93.Phosphate Solubilizing Bacteria (PSB) play important role by enhancing phosphate availability bounded with Al3+ or Fe3+ in acidic soils to oil palm plants through release the inorganic phosphate by enzyme or organic acids solubilization. The aims of this study were to isolate of PSB from oil palm rhizosphere and to conduct a comparative analysis of the solubility inorganic phosphates source by selected PSB. The ability of 15 selected PSB to grow and solubilize aluminum phosphate (AlPO4) and iron phosphate (FePO4) was examined and identified. The highest phospate solubilising efficiency showed K3.1 isolate with phosphate solubilization index 3.2 on NBRIP media. Quantitative analysis revealed that isolate K3.1 solubilized 53.52 mg/mL phosphate in 5 days after being inoculated in AlPO4 containing liquid medium, isolate A4 solubilized 63.45 mg/mL phosphate in 5 days after being inoculated in FePO4 containing liquid medium accompanied by a decrease in pH of the growth medium. Based on the 16s rRNA gene sequence analysis, isolate K3.1 and A.4 were closely related to Burkholderia arboris and Burkholderia gladioli. This potential isolates can be used in order to make oil palm crops more sustainable especially on marginal soil with low pH and less dependent on inorganic P fertilizers.

The predominant oaks of the Maryland Piedmont (Quercus alba and Q velutina) do not occur on serpentine soils. On serpentine sites they are replaced by Q. stellata and Q marilandica. Soil and plant water relations were measured to evaluate water as a limiting factor to distribution of oaks on serpentine soil. May through August soil water potentials of serpentine soils were not lower than adjacent nonserpentine soils. Dawn xylem water potentials of oaks on serpentine soils were similar to nonserpentine oaks during early and midsummer. By late summer, water potentials of the oaks on serpentine sites were higher. Dawn xylem water potentials and soil water potentials were significantly correlated for all species. A significant correlation between xylem water potential and stomatal conductance was found for the oaks on the serpentine soils. No such correlation was found for oaks on nonserpentine soil, but a xylem water potential threshold for stomatal closure was observed. Despite differences in their responses to moisture availability, water does not appear to be the environmental factor responsible for the distributional pattern of these species. INTRODUCTION The occurrence of serpentine soils with their atypical vegetations is a worldwide phenomenon (Walker, 1954). Serpentine soils vary widely in their mineral composition and hence in the edaphic problems encountered by the associated vegetation. Many serpentine soils are high in Mg and have a low Ca:Mg ratio. Toxic levels of Ni, Cr and Co may be present. In addition, one or more mineral nutrients (N, P, K, Mo) may be limiting to plant growth (Proctor and Woodeil, 1975). The physical properties of serpentine soils may also be adverse to plant growth (Proctor and Woodell, 1975). Frequently the soils are severely eroded, shallow and stony. These conditions often result in low soil water availability, which has been hypothesized (Monteferrante, 1973; Miller, 1977) to be an important influence on the vegetation. Whittaker (1954) observed that along a moisture continuum, plant communities of serpentine soils are more xeric than the vegetation of other soils. Few studies have examined the availability of water to plants on serpentine soils. Whittaker (1960) measured soil water in northern California and southern Oregon and found no significant differences between serpentine and nonserpentine soils. Miller (1977) measured soil water parameters (capillary water, permanent wilting percentage and available water) and found no intrinsic differences between a serpentine soil of southeastern Pennsylvania and an adjacent nonserpentine site. However, when he measured the available water in June 1964, there was less available water in the serpentine soil. He concluded that soil moisture and nutrients may have a synergistic effect in limiting the vegetation on serpentine soils. Monteferrante (1973) suggested that, in addition to soil minerals, the shallowness of the soils with their lowered water storage capacity was responsible for the patterns of vegetation. S. N. Turitzen (pers. comm.) studied the diurnal water relations of populations of Smilax rotundifo1ia on and off serpentine soil in southeastern Pennsylvania. He found similar predawn xylem water potentials in both populations, but lower midday xylem water potentials in the serpentine populations. He suggested that differences in exposure accounted for the differences in diurnal water relations. The purpose of this study was to evaluate water relations of oak species found on and off serpentine soils in order to infer if plant moisture stress may be a limiting factor for the oaks on serpentine soils.

A study was undertaken to investigate the occurrence of phosphate solubilizing bacteria (PSB) and nitrogen-fixing bacteria (NFB) from soil samples of Wamena Biological Garden (WbiG). Eleven soil samples were collected randomly to estimate microbial population which used plate count method. The result showed that the microbial population ranged from 5.0x103-7.5x106 cells of bacteria/gram of soil and 5.0x103-1.5x107 cells of bacteria/gram of soil for PSB and NFB respectively. There were 17 isolates which have been identified till genus and species. The isolated microorganism were identified as PSB i.e. Bacillus sp., B. pantothenticus, B. megatherium, Flavobacterium sp., F. breve, Klebsiella sp., K. aerogenes, Chromobacterium lividum, Enterobacter alvei, E. agglomerans, Pseudomonas sp., Proteus sp. and as NFB i.e. Azotobacter sp., A. chroococcum, A. paspalii, Rhizobium sp., and Azospirillum sp.© 2005 Jurusan Biologi FMIPA UNS SurakartaKey words: phosphate- solubilizing bacteria, Nitrogen-fixing bacteria, Wamena Biological Garden

Phosphate Solubilizing Ability and Phylogenetic Diversity of Bacteria from P-Rich Soils Around Dianchi Lake Drainage Area of China

Abstract. Purwanti D, Setyaningsih R, Susilowati A. 2019. Isolation and identification of phosphate solubilizing bacteria from the rhizosphere of rice in organic and non-organic rice fields in Sukoharjo District, Indonesia. Bonorowo Wetlands 9: 108-115. One of the essential nutrients in the soil is phosphate. Phosphate fertilization is often inefficient because phosphate is bound and difficult for plants to absorb. The use of phosphate solubilizing bacteria (PSB) can be used as an alternative to the availability of P in the soil. This study aimed to obtain PSB isolates with high phosphate solubilization ability and identify these PSB isolates molecularly. Soil samples were taken from organic and non-organic rice (Oryza sativa L.) fields in Sukoharjo, Central Java, Indonesia, namely Grogol Village, Menuran Village, and Pondok Village. The research activity was carried out in several stages: isolation of PSB on Pikovskaya medium, characterization and testing of PSB in dissolving P, and Gram staining. PSB colonies with a phosphate solubilization index and showed different colony colors were taken, and then DNA was isolated. DNA isolation was carried out by extraction method using a DNA extraction kit. The gene encoding 16S rRNA amplified the obtained DNA samples. Sequencing was carried out using a genetic analyzer. Data analysis by comparing sequence data on the BLASTN program. Based on the isolation of rhizosphere samples from organic paddy fields, 14 isolates of PSB were obtained, and ten isolates of non-organic paddy were PSB. P13, P1, Q7, and Q8 isolates had the highest P solubilization ability with dissolution index (IP), namely 3.35, 2.13, 1.82, and 1.47. The 16S rRNA gene sequence analysis showed that isolates P1 and P13 had 99% similarity to Acinetobacter sp. ADP1 strain. The isolate Q7 had a 96% similarity with the genus Clavibacter strain NCPPB 382, ??while Q8 had a 99% similarity with Pseudomonas aeruginosa strain PAO1.

Phosphate-solubilizing bacteria (PSB) have the ability to dissolve insoluble phosphate and enhance soil fertility. However, the growth and mineral phosphate solubilization of PSB could be affected by exogenous soluble phosphate and the mechanism has not been fully understood. In the present study, the growth and mineral phosphate-solubilizing characteristics of PSB strain Burkholderia multivorans WS-FJ9 were investigated at six levels of exogenous soluble phosphate (0, 0.5, 1, 5, 10, and 20 mM). The WS-FJ9 strain showed better growth at high levels of soluble phosphate. The phosphate-solubilizing activity of WS-FJ9 was reduced as the soluble phosphate concentration increased, as well as the production of pyruvic acid. Transcriptome profiling of WS-FJ9 at three levels of exogenous soluble phosphate (0, 5, and 20 mM) identified 446 differentially expressed genes, among which 44 genes were continuously up-regulated when soluble phosphate concentration was increased and 81 genes were continuously down-regulated. Some genes related to cell growth were continuously up-regulated, which would account for the better growth of WS-FJ9 at high levels of soluble phosphate. Genes involved in glucose metabolism, including glycerate kinase, 2-oxoglutarate dehydrogenase, and sugar ABC-type transporter, were continuously down-regulated, which indicates that metabolic channeling of glucose towards the phosphorylative pathway was negatively regulated by soluble phosphate. These findings represent an important first step in understanding the molecular mechanisms of soluble phosphate effects on the growth and mineral phosphate solubilization of PSB.

Isolation and screening of phosphate solubilizing bacteria (PSB) seems to become cumbersome using conventional methods, and there may be a chance to lose the more potent isolates during the screening methodology. To overcome these problems, a selective enrichment method has been developed to isolate and screen efficient PSB from diverse sources. Initially, this method includes enrichment of the phosphate solubilizers in the sample followed by selective isolation and screening. Looking into the physico-chemical data, it was revealed that the soil samples from cultivated soil were found to support growth and development of microbial life in general compared to soil samples from non-cultivated stringent environment, which was reflected in number of PSB isolated, or in some samples, PSB may not be obtained. Here, the selective enrichment method developed for isolation of PSB from stringent environmental samples yielded numerically higher number (14) of phosphate solubilizer isolates in comparison to conventional methods (4) from different soil sources. Moreover, isolates obtained following selective enrichment were found consistence for phosphate solubilization in different conditions and for different sources. In contrast to conventional isolation methodologies, the selective enrichment method consistently performed best for PSB isolation particularly from stringent habitats, i.e. mining region where conventional method/s did not perform well.

The present study was conducted to isolate phosphate solubilizing bacteria (PSB) from rhizospheric saline soils of coastal Odisha, India and evaluated their phosphate solubilizing ability. Total four PSB were isolated based on the halo zone formation (solubilizing index 2.63-3.14) on PVK agar medium and were characterized based on biochemical and molecular characteristics as Bacillus subtilis (B1), B. megaterium (B2), Sphingomonas paucimobilis (P2) and Kocuria kristinae (P6). The inorganic phosphate released by PSB ranged from 18.532 to 38.250µg/ml with decreasing the pH PVK broth up to 3.9. Acid phosphatase activity for PSB were recorded 84.237-98.658µmol/min. Glucose was found to be the best carbon source for B. subtilis, Sphingomonas paucimobilis and Kocuria kristinae whereas mannitol for B. megaterium. Optimum acid phosphatase activity was observed for all the four PSB isolates in presence of ammonium sulphate as nitrogen source in PVK broth at 30oC and pH 7.0.