Effect of buffering on the phosphate-solubilizing ability of microorganisms

Native microflora present in the alkaline vertisols and two phosphate-solubilizing bacteria (PSB) isolated from soil using conventional screening media could not release phosphorus from alkaline Indian vertisol soils supplemented with carbon and nitrogen sources. The two PSBs could solubilize both rock phosphate and di-calcium phosphate in unbuffered media but failed to solubilize rock phosphate in buffered media. The organic acids secreted by these PSBs were 20–50 times less than that required to solubilize phosphorus from alkaline soil.     

溶磷菌对4种难溶性磷酸盐溶解能力的初步研究

以4种难溶性磷酸盐为培养基,发现供试菌株溶解这些磷酸盐的特性差异很大,真菌溶磷能力普遍比细菌要高得多。以NO3-为氮源时的溶磷量通常高于以NH4+为氮源时的溶磷量,只有2TCiF2对氟磷灰石及4TCiF6对磷酸铝的溶解能力以NH4+为氮源时较高。大多数菌株较易溶解CaP(氟磷灰石和磷矿粉),其次为AlP(AlPO4),而溶解FeP(FePO4·4H2O的能力都比较弱,只有曲霉2TCiF2具有较强的溶解FeP能力,尤其是当供给NO3-时,溶解FeP的活性比供给NH4+时大幅度提高。欧文氏菌4TCRi22和肠杆菌1TCRi15能大量地溶解氟磷灰石,而两株节杆菌对磷矿粉的溶解能力最强。供试菌株的溶磷作用可能是由于分泌的有机酸与金属离子络合或螯合作用所致,欧文氏菌和肠杆菌溶解难溶性磷过程中,非有机酸物质可能在起主要作用。     

Critical Evaluation of Phosphate Solubilizing Pseudomonads Isolated from a Partially Recultivated Potash Tailings Pile

Ten phosphate solubilizing pseudomonads isolated from a partially recultivated potash tailings pile in Germany were characterized and tested for their potential to assist in the ongoing recultivation process. Despite fertilization, the plants which are grown for recultivation show phosphate deficiency symptoms, and therefore the isolates are intended to be used as biofertilizer inoculants. On agar plates incubated at five different temperatures, some of the strains showed a temperature-dependent ability to solubilize tricalcium phosphate, while others performed the same at any given temperature. In liquid medium, the isolates solubilized between 271 and 730 μg ml(-1) of phosphate from tricalcium phosphate. Both the weakest (designated S10) and the strongest solubilizing strain (S06) were further tested for their viability during solubilization. In an assay over the course of 1 week, both strains released their maximum amount of phosphate after 2-4 days. At that later point of time, however, viable cells of isolate S06 were no longer detectable, whereas the weaker strain S10 could be cultured after 1 week in broth. Taking all in vitro observations into account, the usability of the isolates as biofertilizers is critically discussed regarding both the in situ conditions on the tailings pile and the lowered viability due to the excess production of organic acids.     

Plant Growth-Promoting Effect of the Chitosanolytic Phosphate-Solubilizing Bacterium Burkholderia gladioli MEL01 After Fermentation with Chitosan and Fertilization with Rock Phosphate

Phosphate-solubilizing bacteria (PSB) are important plant growth-promoting rhizobacteria that can increase soil fertility through the solubilization of insoluble inorganic phosphate and organophosphorus. In this study, a PSB, Burkholderia gladioli MEL01, was isolated and identified from rice–wheat rotation rhizosphere soil. MEL01 had an excellent phosphate-solubilizing capacity (reaching 107.69 mg/L) toward insoluble inorganic phosphate rock phosphate. HPLC analysis revealed that the mechanism of phosphate solubilization of MEL01 was probably due to secreted oxalic acid and gluconic acid transformation of phosphate from insoluble to soluble. MEL01 also exhibited 4030 U/L specific chitosanase activity when cultured with chitosan fermentation medium. Interestingly, the chitosan hydrolysis product chitooligosaccharide could significantly enhance the MEL01 phosphate-solubilizing capacity. Pot experiments showed that MEL01 chitosan medium fermentation liquor (MCMFL) could promote improvement of soil available phosphorus and pakchoi growth when supplemented with phosphate rock phosphate as the phosphate fertilizer. In addition, pot experiments demonstrated that MCMFL could also promote the growth of wheat, which could decrease the amount of compound fertilizer used. Microbial diversity analysis showed that the genera Pseudomonas, Burkholderia, Mycoplana, and Cellvibrio were enriched, which might participate in synergetic phosphate solubilization. Therefore, after fermentation with chitosan and fertilization with rock phosphates, MEL01 has potential as a phosphate biofertilizer in ecological agricultural production.

     

Novel P-Solubilizers from Calcium Bound Phosphate Rich Pine Forest of Lower Himalaya

Phosphorus, an essential element for life, is continuously depleting from soils and thus demands sustainable management particularly in agriculture and forestry. Inorganic P constitutes the major proportion as tricalcium phosphate in soils of lower Himalayan region of Pakistan. We sampled these soils and screened for P-solubilizing microbes. A range of culturable microbial community (bacteria and fungi) was isolated and molecularly characterized which make the P available from mineral phosphates. There was an increase in abundance of phosphate solubilizing bacteria (PSB) at a 6-inch depth of the pine rhizosphere compared to the surface soil samples. Moreover, the isolates from lower Himalaya have higher abundance and better efficiency to solubilize the inorganic P than the ones from non-Himalaya. Most likely the P-solubilization done by our P-solubilizing microbes is via acidification as we observed the decrease in pH of the medium of microbial growth. Furthermore, the majority of isolated PSB belong to gammaproteobacterial class of Gram negative bacteria. Most interestingly, 13% of our isolated PSB were psychrotolerant (physiologically active at cold environment, i.e., 4°C) and able to solubilize inorganic P as efficiently as at ambient temperature. This study is unique in reporting the P-solubilizing microbes, particularly the psychrotolerant bacterial strains, of Lower Himalaya. Therefore the isolated bacterial and fungal strains have potential and may serve as biofertilizers in the region to increase the P availability in soils.     

Characterization of phosphobacteria isolated from eutrophic aquatic ecosystems

Phosphobacteria are able to enhance phosphorus availability in soil and improve crop yields. To develop such biofertilizers, 14 predominant phosphobacteria were isolated from eutrophic aquatic ecosystems. Molecular identification and phylogenetic analysis revealed three groups among the nine isolates of inorganic phosphate-solubilizing bacteria (IPSB): IPSBl and IPSB2 belonged to the actinobacteria and flavobacteria, respectively, and the other seven belonged to the γ-proteobacteria. Among five isolates of organic phosphorus-mineralizing bacteria (OPMB), two groups were present: OPMB1 and OPMB3 belonged to the β-proteobacteria, while the other three belonged to the γ-proteobacteria. The IPSB isolates released 62.8–66.7 mg P 1⁻¹ from tricalcium phosphate under shaking conditions, and 26.8 to 43.7 mg P 1⁻¹ under static conditions; the OPMB strains released 23.5–30.2 mg P 1⁻¹ from lecithin under shaking conditions, and 16.7–27.6 mg P 1⁻¹ under static conditions. To the best of our knowledge, this is the first report indicating that IPSBl (designated Aureobacterium resistents) as a tricalcium phosphate-solubilizing bacterium and OPMB1 and OPMB3 (designated Acidovorax temperans and Achromobacter xylosoxidans, respectively) are lecithin-mineralizing bacteria. This investigation demonstrated that a eutrophic aquatic ecosystem is a selective source of phosphobacteria and the screened phosphobacteria are a potential alternative to the development of biofertilizers.     

一株高效解无机磷细菌BS06的鉴定及其解磷能力分析

【目的】对一株来源于广西甘蔗根际土壤的高效解无机磷细菌BS06的分类和解磷能力进行探讨,以期为解磷微生物在广西甘蔗生产上的开发和应用提供理论依据。【方法】通过形态观察、生理生化测定及16S rRNA基因序列同源性分析,进一步结合种特异的recA基因序列分析对解磷菌BS06进行分类鉴定;通过改变无机磷培养基中的碳源、氮源对菌株解磷能力的影响,分析菌株的解磷特性;通过盆栽试验了解菌株对甘蔗品种粤糖00236、桂糖28磷素吸收的影响。【结果】分类鉴定结果表明菌株BS06属于洋葱伯克霍尔德菌(Burkholderia cepacia);菌株在以乳糖为碳源条件下具有较强的解磷能力,其发酵液中水溶性磷含量为262.71 mg/L;在以硝酸钠为氮源条件下有较强解磷能力,其发酵液中水溶性磷含量达到305.85 mg/L;接种BS06菌株显著促进甘蔗组培苗的生长并提高甘蔗植株的含磷量。【结论】解磷细菌BS06具有较大的开发利用潜力。     

Marine Actinobacteria Showing Phosphate-Solubilizing Efficiency in Chorao Island, Goa, India

The occurrence and distribution of an actinobacteria group of bacteria capable of dissolving insoluble phosphates were investigated in this study in marine environments, especially in sediments of Chorao Island, Goa Province, India. A total of 200 bacterial isolates of actinobacteria was isolated. All isolates were screened for phosphate-solubilizing activity on Pikovskaya’s agar. Thirteen different isolates exhibiting maximum formation of halos (zone of solubilization) around the bacterial colonies were selected for quantitative estimations of P-solubilization. Quantitative estimations for P-solubilization were analyzed for up to 10 days at intervals of 24 h. Maximum solubilization from 89.3 ± 3.1 to 164.1 ± 4.1 μg ml^?1 was observed after 6 days of incubation in six of all isolates, while the isolate NII-1020 showed maximum P-solubilization. The increase in solubilization coincided with the drop in pH. Many of these species showed wide range of tolerance to temperature, pH, and salt concentrations. Further, 16S rRNA gene sequence analyses were carried to identify the bacterial groups which are actively solubilized phosphate in vitro. Gene sequencing results reveal that all isolates were clustered into six different actinobacterial genera: Streptomyces, Microbacterium, Angustibacter, Kocuria, Isoptericola, and Agromyces. The presence of phosphate-solubilizing microorganisms and their ability to solubilize phosphate were indicative of the important role played by bacteria in the biogeochemical cycle of phosphorus and the plant growth in coastal ecosystems.     

Tricalcium phosphate solubilizing abilities of Trichoderma spp. in relation to P uptake and growth and yield parameters of chickpea (Cicer arietinum L.)

Nine isolates of Trichoderma spp. were investigated for their ability to solubilize insoluble phosphate in Pikovskaya's broth and were compared with an efficient phosphate-solubilizing bacterium Bacillus megaterium subsp. phospaticum PB that was used as the reference strain. All 9 Trichoderma isolates were found to solubilize insoluble tricalcium phosphate to various extents. Trichoderma viride (TV 97) (9.03 microg x mL(-1)), Trichoderma virens (PDBCTVs 12) (9.0 microg x mL(-1)), and Trichoderma virens (PDBCTVs 13) (8.83 microg x mL(-1)) solubilized 70% of that solubilized by the reference strain Bacillus megaterium (12.43 microg x mL(-1)). Pot culture and field evaluations with Trichoderma harzianum (PDBCTH 10), Trichoderma viride (TV 97), and Trichoderma virens (PDBCTVs 12) using chickpea (Cicer arietinum L.) 'Annegeri-1' as the test plant and rock phosphate as the phosphorus source showed significantly increased P uptake in plants treated with Trichoderma harzianum (PDBCTH 10) followed by Trichoderma virens (PDBCTVs 12) and Trichoderma viride (TV 97). Inoculation of Trichoderma spp. also showed increased growth and yield parameters of chickpea compared with the uninoculated controls under both glasshouse and field conditions.     

一株嗜松青霉JP-NJ4的解磷特性

【目的】土壤中磷素供应不足是造成马尾松林地力衰退的原因之一。本研究对前期从马尾松根际土样中分离筛选出的一株解磷能力较强的嗜松青霉JP-NJ4的解无机磷及解有机磷能力进行探讨。【方法】探究嗜松青霉JP-NJ4对4种无机磷源及2种有机磷源的降解能力,并对其分泌的有机酸和酶类进行测定,对其解磷特性进行初步分析。【结果】表明JP-NJ4菌株可在4种不同无机磷源的培养基中生长,其中对磷酸钙[Ca3(PO4)2]的解磷效果最好,对4种磷源的解磷能力大小为:磷酸钙磷酸铝磷酸氢钙磷酸铁;其分泌的有机酸种类主要为葡萄糖酸、草酸及丙二酸;JP-NJ4菌株的磷酸酶活性较高,并具有一定的植酸酶活性;同时对草甘膦具有较好的生物降解功能,降解率达49.6%。【结论】嗜松青霉JP-NJ4解磷能力受磷源的结构组成影响,且解磷能力与发酵液pH值呈负相关关系;该菌株分泌的葡萄糖酸和草酸对磷酸钙及磷酸铝的溶解效果较明显。本研究供试菌株嗜松青霉JP-NJ4具有良好的解磷功能,在作为林业生物菌肥方面具有极大的应用潜力。     

Genetic diversity of phosphate-solubilizing peanut (Arachis hypogaea L.) associated bacteria and mechanisms involved in this ability

In this study, attempts were made to analyze mechanisms involved in the bacterial phosphate-solubilizing ability of peanut isolates. Bacteria were taxonomically identified by analysis of 16S rDNA sequence. Levels of soluble P released by the isolates in unbuffered or buffered with Tris–HCl or MES NBRIP-BPB medium as well as the production of D-gluconic acid were determined in their culture. Presence of two of the genes encoding the cofactor PQQ of GDH enzyme was analyzed in the genome of this bacterial collection. 16S rDNA sequence analysis indicated that isolates belong to genera Serratia, Enterobacter, Pantoea, Acinetobacter, Bacillus and Enterococcus. All bacteria showed ability to solubilize tricalcium phosphate either in unbuffered or buffered medium. Nevertheless, addition of buffer solutions reduced levels of Pi liberated by the isolates. Although almost all isolates produced detectable amounts of D-gluconic acid, no correlation with levels of P soluble released were observed. The presence of pqqE and pqqC genes was detected only in Gram negative bacteria. It was concluded from this study that the mechanism involved in phosphate solubilization is organic acids production and, presence of pqq genes in all Gram negative bacteria analyzed encourages to confirm their role in bacterial phosphate solubilizing ability as well to identify genes involved in this PGP trait in Gram positive bacteria.     

滨海盐碱地根际溶磷细菌磷素转化特征

为探讨溶磷细菌对土壤磷素的转化效果,提高黄河三角洲盐碱地土壤肥力。从黄河三角洲盐地碱蓬根际土壤中选取一株高效溶磷细菌RPB03,采用单因子实验,探究不同环境因子对RPB03菌株溶磷效果的影响。结果表明：RPB03菌株为Pantoea vagans,隶属泛菌属。在密闭培养方式下,溶磷菌RPB03菌株在较高的盐度、温度和碱性条件下,溶磷量皆达到300 mg/L以上,溶磷能力良好。盆栽实验结果表明,该菌可有效促进土壤中无效态磷向有效态磷的转化（有效磷含量从0.029 mg/kg提升至0.043 mg/kg）。研究表明,RPB03菌株是一株耐盐碱性较强的高效溶磷细菌,适合在黄河三角洲盐碱地中生存,且其存在对提升黄河三角洲盐碱土壤有效磷含量具有促进作用。     

Mechanism of insoluble phosphate solubilization by Pseudomonas fluorescens RAF15 isolated from ginseng rhizosphere and its plant growth-promoting activities

Aims:  To investigate the mechanism of insoluble phosphate (P) solubilization and plant growth-promoting activity by Pseudomonas fluorescens RAF15.
Methods and Results:  We investigated the ability of Ps. fluorescens RAF15 to solubilize insoluble P via two possible mechanisms: proton excretion by ammonium assimilation and organic acid production. There were no clear differences in pH and P solubilization between glucose-ammonium and glucose-nitrate media. P solubilization was significantly promoted with glucose compared to fructose. Regardless of nitrogen sources used, Ps. fluorescens RAF15 solubilized little insoluble P with fructose. High performance liquid chromatography analysis showed that Ps. fluorescens RAF15 produced mainly gluconic and tartaric acids with small amounts of 2-ketogluconic, formic and acetic acids. During the culture, the pH was reduced with increase in gluconic acid concentration and was inversely correlated with soluble P concentration. Ps. fluorescens RAF1 showed the properties related to plant growth promotion: pectinase, protease, lipase, siderophore, hydrogen cyanide, and indoleacetic acid.
Conclusion:  This study indicated that the P solubility was directly correlated with the organic acids produced.
Significance and Impact of the Study:  Pseudomonas fluorescens RAF15 possessed different traits related to plant growth promotion. Therefore, Ps. fluorescens RAF15 could be a potential candidate for the development of biofertilizer or biocontrol agent.     

一株金黄蓝状菌解磷特性及其对毛竹的促生效应

为深入了解毛竹根际微生物金黄蓝状菌(JXBR04)的解磷特性并评价其促生效应,采用液体发酵培养法研究碳源、氮源、pH、装液量及盐离子等因素对金黄蓝状菌解磷能力的影响,以及该菌株对不同难溶性磷酸盐的溶解能力,并应用温室盆栽法研究该菌株对毛竹幼苗生长的促进效应.结果表明: 金黄蓝状菌(JXBR04)分别在碳源为蔗糖、氮源为酵母粉、初始pH值3.5、装液量1/5或2/5、盐离子浓度为0 或1.0 g·L^-1时解磷能力最强;对Ca₃(PO₄)₂、CaHPO₄、FePO₄均具有较好的解磷作用,其中对CaHPO₄表现最佳,达1304.04 mg·L^-1.施用金黄蓝状菌(JXBR04)菌剂180 d后显著提高了毛竹实生苗根际土壤养分和植株体内磷含量,且地径、苗高及生物量分别比对照增长了28.1%、28.3%和51.5%.可见,金黄蓝状菌具有成为我国南方酸性土壤毛竹林环境友好型生物肥料的潜力.     

Solubilization of insoluble inorganic phosphates by a novel temperature-, pH-, and salt-tolerant yeast, <Emphasis Type="Italic">Rhodotorula</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> PS4, isolated from seabuckthorn rhizosphere,growing in cold desert of Ladakh,India

The study was aimed to develop biofertilizer solubilizing inorganic phosphates for region experiencing temperature, pH and salt stressed conditions. A yeast strain PS4, which was temperature-, pH- and salt-tolerant and capable of solubilizing insoluble inorganic phosphate was isolated from rhizosphere of seabuckthorn (Hippophae rhamnoides L.), growing in the Indian Trans-Himalaya. Based on morphological, biochemical, whole cell FAME analysis and molecular characterization, strain PS4 was identified as Rhodotorula sp. The soluble phosphate production under optimal conditions at pH 7 and 30°C was 278.3 mg l⁻¹. Strain PS4 showed ability to solubilize insoluble phosphate under different stress conditions viz. 5–40°C temperature, 1–5% salt concentration and 3–11 pH range. Soluble phosphate production from Ca₃(PO₄)₂ under combined stress conditions at extreme values of temperature, pH and salt concentration showed 81.6–83.2% reduction as compare to optimal conditions after 5 days incubation. The strain solubilize Ca₃(PO₄)₂ to a great extent than FePO₄ and AlPO₄. The solubilization of insoluble phosphate was associated with drop in pH of the culture media. Inoculation of tomato seedling with the strain increased fruit yield, roots and shoot length. Rhodotorula sp. PS4 with phosphate-solubilizing ability under stress conditions appeared to be attractive for exploring their plant growth-promoting activity towards the development of microbial inoculants in stressed region.     

Variable root:shoot ratios and plant nitrogen concentrations discourage using just aboveground biomass to select legume service crops

Plant and Soil - The efficiency test of phosphate-solubilizing bacteria (PSB) biofertilizer had two objectives: (i) to study the effect of the use of PSB on phosphate solubilization of the Takhli...     

丢糟和磷矿粉高温堆肥中耐高温解磷菌的筛选及性能分析

为了解决高温极端环境下的磷素溶出问题,从高温堆肥中分离出具有耐高温能力的解磷微生物。该研究利用无机磷选择培养基,从添加磷矿粉和丢糟的高温堆肥样品中,分离筛选出5株耐高温解磷菌(细菌为GDB1-2;真菌为GDF1-3),并对这5株菌株进行形态学和分子生物学鉴定及解磷能力分析。研究结果表明,筛选获得的解磷菌株分别为枯草芽孢杆菌(Bacillus subtilis)、地衣芽孢杆菌(Bacillus licheniformis)、多枝横梗霉(Lichtheimia ramosa)、烟曲霉(Aspergillus fumigatus)及构巢曲霉(Aspergillus nidulans)。该5株菌具有较好的耐高温和耐pH性能,各菌株耐高温范围为40-60℃,在50℃条件下其解磷能力均达到最大值,当初始pH 在5-9范围时各菌株均能保持一定的解磷能力。此外,通过解磷曲线发现各菌株的最高解磷量范围在136.85-174.33 μg/mL。该研究结果为后续开发高温环境微生物资源提供了素材,具有良好的应用推广前景。     

Optimizations of particle size and pulp density for solubilization of rock phosphate by a microbial consortium from activated sludge

Microbial solubilization of rock phosphate is getting more and more attention recently. However, the microorganisms used in previous studies were mostly single or known species, and seldom studies focused on the mixed microorganisms or microbial consortia from natural environments. In this study, a microbial consortium taken from activated sludge was used to solubilize two different mid-low-grade rock phosphates. The results showed that the microbial consortium could effectively solubilize the rock phosphates in National Botanical Research Institute’s phosphate growth medium and released soluble phosphorus in the broth. The biomass increased gradually, whereas the pH decreased sharply during the solubilizing process. The maximum phosphorus solubilization was recorded at particle size of 150?µm. Higher or lower than this optimal particle size, the phosphorus solubilization decreased. The phosphorus solubilization gradually decreased with a larger pulp density from 1 to 5%, and the optimal pulp density was 1%. The solubilization level of microbial consortium varied with different rock phosphates. The results revealed that the soluble phosphorus released from high-silicon ore was higher than which from high-magnesium ore. A strong positive correlation between biomass and phosphorus solubilization in the broth was observed from regression analysis results, and the phosphorus solubilization also had a significant negative correlation with pH in the broth.     

Isolated Phosphate-Solubilizing Soil Bacteria Promotes In vitro Growth of Solanum tuberosum L.

The capacity of four bacterial strains isolated from productive soil potato fields to solubilize tricalcium phosphate on Pikovskaya agar or in a liquid medium was evaluated. A bacterial strain was selected to evaluate in vitro capacity of plant-growth promotion on Solanum tuberosum L. culture. Bacterial strain A3 showed the highest value of phosphate solubilization, reaching a 20 mm-diameter halo and a concentration of 350 mg/l on agar and in a liquid medium, respectively. Bacterial strain A3 was identified by 16S rDNA analysis as Bacillus pumilus with 98% identity; therefore, it is the first report for Bacillus pumilus as phosphate solubilizer. Plant-growth promotion assayed by in vitro culture of potato microplants showed that the addition of bacterial strain A3 increased root and stems length after 28 days. It significantly increased stem length by 79.3%, and duplicated the fresh weight of control microplants. In this paper, results reported regarding phosphorus solubilization and growth promotion under in vitro conditions represent a step forward in the use of innocuous bacterial strain biofertilizer on potato field cultures.Key words: Bacillus sp., phosphorus soluble, Pikovskaya agar, potato rhizosphere, plant growth promoting rhizobacteria     

Effectiveness of phosphate and potash rocks with <Emphasis Type="Italic">Acidithiobacillus</Emphasis> on sugarcane yield and their effects on soil chemical attributes

Experiments were conducted to evaluate the effectiveness of biofertilizers produced from phosphate and potash rocks mixed with sulfur inoculated with Acidithiobacillus oxidizing bacteria on sugarcane yield and their effects on some chemical attributes of a Brazilian tableland soil. The experiment was arranged in a completely randomized factorial design 2 × 4 × 3 + 1, with four replicates. Two varieties of sugarcane, four rates of three sources of P and K (apatite + biotite, P + K biofertilizers with Acidithiobacillus, and soluble fertilizers—triple superphosphate and potassium chloride) were tested. A control without P and K fertilization was applied. A significant reduction in soil pH was observed with biofertilizers, especially when applied at higher rates, although no harmful effect on sugarcane yield was observed. Available P and K and exchangeable Ca and Mg increased with biofertilizer application compared to mineral fertilizers and P and K rocks. Biofertilizers may be used as an alternative source of P and K for sugarcane grown in soils with low available P and K.