Xanthomonas campestris, a novel stress tolerant, phosphate-solubilizing bacterial strain from saline–alkali soils

A total of 198 bacterial strains were isolated from various niches of saline–alkali soils, out of which 85 strains were able to solubilize phosphate on plates at 30 °C. The strain RMLU-26, identified as Xanthomonas campestris, was the most efficient with its ability to solubilize P, subjected to N-methyl-N′-nitro-N-nitrosoguanidine (NTG) for development of mutants. The P solubilizing ability of X. campestris is reported for the first time. The wild type and mutant strains of X. campestris revealed a differential response to various stress factors (high pH, temperature, and salt concentration). The mutant strain revealed maximum P solubilization (67.1%) at 30 °C and pH 8.0 while the wild type strain showed maximum solubilization (41.9%) at 35 °C and pH 7.0. Percent P₂O₅ solubilization by both strains revealed a steep decline in tricalcium phosphate solubilization with an increase in NaCl concentration from 0.5 to 10% along with a concomitant drop in pH of the medium from 8.0 to 4.5 in wild type and 4.0 in mutant strain. However, a 1.5- to 2-fold increase in ‘P’ solubilization was observed in the mutant strain when compared to the wild type strain in the presence of NaCl. The overall improved tolerance of the strains to alkalinity and salinity could be due to accumulation and/or secretion of specific solute (xanthan).     

Characterization of plant growth-promoting rhizobacterium <Emphasis Type="Italic">Exiguobacterium</Emphasis> NII-0906 for its growth promotion of cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis>)

A phosphate solubilizing and antagonistic bacterial strain, isolated from a Western Ghat forest soil in Kerala province, India (designated as NII-0906), showed cold tolerance and grew from 10 to 37°C (optimum temperature 30°C). It was a Gram-positive, rod shaped, 0.8–1.6 μm in size, and exhibited tolerance to a wide pH range (5–12; optimum 7.0) and salt concentration up to 7% (w/v). The isolate showed maximum similarity with Exiguobacterium marinum TF-80^T based on 16S rRNA analysis. It solubilized tricalcium phosphate under in vitro conditions. The phosphate solubilization was estimated along a temperature range (5–40°C), and maximum activity (84.7 μg mL⁻¹ day⁻¹) was recorded at 30°C after 10 days of incubation. The phosphate solubilizing activity coincided with a concomitant decrease in pH of the medium. The isolate also exhibited antifungal activity against phytopathogenic fungi in Petri dish assays and produced siderophore and hydrogen cyanide. The strain’s plant growth promotion properties were demonstrated through a cowpea-based bioassay under greenhouse conditions. The bacterial inoculation resulted in significant increment in plant root, stem and as well as in plant biomass. Further, scanning electron microscopic study revealed the root colonization in cowpea. These results could offer potential perspective for the strain to be used as plant growth-promoting rhizobacteria, which could be used as an inoculant for regional crops.     

Effect of Abiotic Stress on Phosphate Solubilization by Biocontrol Fungus <Emphasis Type="Italic">Trichoderma</Emphasis> sp.

This study was undertaken to explore the role of Trichoderma sp. in phosphate (P) solubilization and antagonism against fungal phytopathogens. All fungal isolates (SE₆, KT₆, KT₂₈, and BRT₁₁) and a standard culture of T. harzianum (Th-std) were able to antagonize two fungal phytopathogens (Sclerotium rolfsii and Rhizoctonia solani) of chickpea (Cicer arietinum L.) wilt complex. Transmission electron microscopic studies (TEM) further confirmed ultra-cytological changes in the sclerotia of S. rolfsii parasitized by Trichoderma sp. All fungal cultures exhibited production of NH₃ and siderophore, but only BRT₁₁, SE₆, and Th-std could produce HCN. Among all the cultures tested, isolate KT₆ was found to be most effective for solubilization of ferric phosphate releasing 398.4 μg ml⁻¹ phosphate while isolates BRT₁₁ and SE₆ showed more potential for tricalcium phosphate (TCP) solubilization releasing 449.05 and 412.64 μg ml⁻¹ phosphate, respectively, in their culture filtrates. Part of this study focused on the influence of abiotic stress conditions such as pH, temperature, and heavy metal (cadmium) on phosphate (TCP) solubilizing efficiency. Two selected cultures KT₆ and T. harzianum retained their P solubilizing potential at varying concentrations of cadmium (0–1000 μg ml⁻¹). Isolate KT₆ and standard culture of T. harzianum released 278.4 and 287.6 μg ml⁻¹ phosphate, respectively, at 1000 μg ml⁻¹cadmium. Maximum solubilization of TCP was obtained at alkaline pH and at 28°C temperature. Isolate BRT₁₁ was found most alkalo-tolerant releasing 448.0 μg ml⁻¹ phosphate at pH 9.     

Stabilization of dextransucrase from <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> NRRL B-640

Phosphate solubilizing yeast (PSY) were isolated from rhizosphere, non-rhizosphere and fruits from Bhavnagar district. The potential of 25 yeasts were analyzed on the basis of phosphate solubilizing zone to growth on solid medium denoted as solubilization index (SI) which ranged from 1.10 to 1.50. Among 25 yeast isolates, 6 yeast belonging to genus Saccharomyces (2), Hansenula, Klockera, Rhodotorula and Debaryomyces exhibited highest SI (1.33–1.50) were further examined for in vitro tricalcium phosphate (TCP) and low grade rock phosphate (RP) solubilization. TCP proved superior to RP with all the yeasts. Within low grade RPs tested, except isolate Y5, all isolates showed maximum solubilization with Hirapur RP (HRP) ranging from 7.24 to 19.30 mg% P₂O₅. Among six PSY screened, Debaryomyces hansenii showing maximal HRP solubilization was chosen for further physiological studies. Maximum HRP solubilization was expressed in following condition: pH optima 7.0, temperature optima 28°C and optimal period of incubation were 15 days. Acidic pH of the spent media was a constant feature in all the cases. No correlation could be established between final acidity produced by yeasts and the quantity of phosphate liberated.     

Community of extremely halophilic bacteria in historic Dagong Brine Well in southwestern China

The Dagong Brine Well, the earliest man-made brine well in record, located in Zigong, Sichuan, China, had the construction and facilities mainly made of bamboo, wood and stone that are eroded easily by halophiles colonizing in the brine of the well. To better preserve this historic placemark, we initiated an investigation on the microbial community and the diversity of the halophilic bacteria in the brine of Dagong well for the first time. A total of 112 aerobic halophile strains were isolated and characterized according to their phenotype, cellular fatty acids composition and 16S rRNA. Furthermore, the intra specific phylogeny of closely related strains was also screened by PCR fragment length polymorphism of 16S–23S ribosomal RNA intergenic spacer regions (ISR). The result showed that the halophilic isolates found in current study were closely related to the following genera: Planococcus, Halomonas, Halobacillus, Oceanobacillus and Virgibacillus, a lineage of the domain Bacteria. Halomonas and Halobacillus were the dominant genera as represents 27% and 40% of the total isolates, respectively. The distribution of other genera was as follows: Oceanobacillus (18%), Virgibacillus (10%) and Planococcus (5%). The ISR analysis disclosed variation of banding pattern in some isolates related to Oceanobacillus and Halobacillus which was observed with phenotypic and physiological characterizations as well. It was clear that these halophiles have adapted to the special man-made hypersaline environment by the basic physiological evolution during phylogenesis and thus resulted in phenotypic and genotypic diversity in the Dagong Brine Well.     

Growth Enhancement of Chickpea in Saline Soils Using Plant Growth-Promoting Rhizobacteria

Bacterial isolates with the ability to tolerate salinity and plant growth-promoting features were isolated from the saline areas of Gujarat, India, that is, Bhavnagar and Khambat. A total of 176 strains of rhizobacteria were isolated out of which 62 bacterial strains were able to tolerate 1 M NaCl. These were then further studied for their potential plant growth-promoting rhizobacteria characteristics like phosphate solubilization, siderophore production, and IAA production. Twenty-eight isolates of the 62 strains showed good tricalcium phosphate solubilization in solid medium in the range of 9–22 mm and 15 isolates showed good phosphate solubilization in liquid medium in the range of 9–45 μg/ml. Siderophore production was checked in all 15 isolates, and 13 were screened out that produced the hydroxamate type of siderophore in the range of 11–50 mM. Among the 13 isolates, 10 were able to produce indole acetic acid in the range of 10–26 μg/ml after 72 h of incubation. Pot trials were carried out on chickpea under 300 mM NaCl stress using the best five isolates. Plants inoculated with MSC1 or MSC4 isolates showed an increase in the parameters that evaluate plant growth when compared to uninoculated controls. Strains MSC1 and MSC4 were identified as Pseudomonas putida and Pseudomonas pseudoalcaligens, respectively, according to sequence analysis of the 16S rRNA gene.     

Phosphate solubilization potential and stress tolerance of rhizobacteria from rice soil in Northern Thailand

Plant growth-promoting rhizobacteria (PGPR) are known to influence plant growth by various direct or indirect mechanisms. A total of 216 phosphate-solubilizing bacterial isolates were isolated from different rice rhizospheric soil in Northern Thailand. These isolate were screened in vitro for their plant growth-promoting activities such as solubilization of inorganic phosphate, ammonia (NH₃), catalase and cell wall-degrading enzyme activity. It was found that 100% solubilized inorganic phosphate, 77.77% produced NH₃ and most of the isolates were positive for catalase. In addition, some strains also produced cell wall-degrading enzymes such as protease (7%), chitinase (1%), cellulase (3%) and β-glucanase (3%), as evidenced by phenotypic biochemical test and quantitative assay using spectrophotometry. The isolates could exhibit more than two or three plant growth-promoting (PGP) traits, which may promote plant growth directly or indirectly or synergistically. Part of this study focused on the effect of NaCl, temperature, and pH on a specific the bacterial isolate Acinetobacter CR 1.8. Strain CR 1.8 was able to grow on up to 25% NaCl, between 25 and 55°C, and at pH 5–9. Maximum solubilization of tricalcium phosphate and aluminium phosphate was obtained at neutral pH, and 37°C. Strain CR 1.8 had protease activity but no cellulase, β-glucanase and cellulase activities.     

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.     

Phosphorus Recovery from Phosphate Rocks Using Phosphate-Solubilizing Bacteria

In this paper, we are presenting a biological process to recover phosphorus by solubilizing low-grade phosphate rocks. To this end, the efficiency of different phosphate-solubilizing microorganism (PSM) species for solubilizing P from phosphate rocks using both pure cultures and associations. Nutritional conditions, phosphate rock concentrations, and reactor designs were tested. The genus Bacillus, especially Bacillus megaterium (ATCC 14581), was found to be the most promising PSM for solubilizing P. Production of organic acids and acidic pH values were shown to be directly related to P solubilizing. However, associations between tested microorganisms did not significantly enhance process efficiency. We conclude that nutritional factors of the medium are important to solubilization, and lower phosphate rock concentrations lead to better solubilization. The Air Lift reactor was promising for B. megaterium (ATCC 14581), but adaptations are needed for further tests.     

Phosphate solubilization by <Emphasis Type="Italic">Rhizobium</Emphasis> strains

Forty-six Rhizobium isolates from legume root and stem nodules were examined for their phosphate-solubilizing ability on Pikovskaya’s agar medium. Rhizobium isolates from root nodules of Cassia absus, Vigna trilobata and three strains from Sesbania sesban showed zone of tricalcium phosphate (TCP) solubilization. The isolate from C. absus showed maximum solubilization (620 μg/ml) after 12 d of incubation, while the Rhizobium sp. strain 26 (from S. sesban) showed the least amount (150 μg/ml) of phosphate solubilization. Among the carbon sources tested for their ability to solubilize TCP, maximum solubilization (620 μg/ml) was observed in glucose by Rhizobium isolate from C. absus. Phosphate solubilization increased with increase in glucose concentration steeply up to 2% and slowly above this concentration in four isolates. Among the nitrogen sources tested, maximum solubilization (620 μg/ml) was observed in ammonium sulphate by Rhizobium isolate from C. absus.     

Phosphate solubilization by Penicillium spp. isolated from soil samples of Indian Himalayan region

A total of 246 fungal isolates representing 36 genera and 72 species were isolated from the soil samples collected from Indian Himalayan region. Twenty-one species belonged to the genus Penicillium alone. All the Penicillium species were screened for phosphate solubilizing activity on Pikovskaya agar at 21 °C. Eight species of Penicillium, exhibiting formation of halos (zone of solubilization) around the fungal colonies in qualitative plate assays, were selected for quantitative estimations. In quantitative estimations that were conducted upto day 30 (at 3 days interval), seven species of Penicillium brought maximum solubilization after day 15, while P. oxalicum showed maximum solubilization after day 21 of incubation. The increase in solubilization coincided with decrease in pH of the broth. Acid phosphatase activity was 1.5–2.0 times higher in comparison to alkaline phosphatase. Many of these species showed wide range of tolerance for temperature, pH and salt concentration.     

Increase in pH stimulates mineralization of ‘native’ organic carbon and nitrogen in naturally salt-affected sandy soils

Large amounts of terrestrial organic C and N reserves lie in salt-affected environments, and their dynamics are not well understood. This study was conducted to investigate how the contents and dynamics of ‘native’ organic C and N in sandy soils under different plant species found in a salt-affected ecosystem were related to salinity and pH. Increasing soil pH was associated with significant decreases in total soil organic C and C/N ratio; particulate (0.05–2 mm) organic C, N and C/N; and the C/N ratio in mineral-associated (<0.05 mm) fraction. In addition, mineral-associated organic C and N significantly increased with an increase in clay content of sandy soils. During 90-day incubation, total CO₂-C production per unit of soil organic C was dependent on pH [CO₂-C production (g kg⁻¹ organic C) = 22.5 pH – 119, R ² = 0.79]. Similarly, increased pH was associated with increased release of mineral N from soils during 10-day incubation. Soil microbial biomass C and N were also positively related to pH. Metabolic quotient increased with an increase in soil pH, suggesting that increasing alkalinity in the salt-affected soil favoured the survival of a bacterial-dominated microbial community with low assimilation efficiency of organic C. As a result, increased CO₂-C and mineral N were produced in alkaline saline soils (pH up to 10.0). This pH-stimulated mineralization of organic C and N mainly occurred in particulate but not in mineral-associated organic matter fractions. Our findings imply that, in addition to decreased plant productivity and the litter input, pH-stimulated mineralization of organic matter would also be responsible for a decreased amount of organic matter in alkaline salt-affected sandy soils.     

Microbial population dynamics, especially stress tolerant Bacillus thuringiensis, in partially anaerobic rice field soils during post-harvest period of the Himalayan, island, brackish water and coastal habitats of India

Population ( × 10⁷ c.f.u./g dr. soil) of the aerobic (30.5–154.1) and anaerobic (5.9–91.4) heterotrophic, aerobic (24.0–56.0) and anaerobic (2.4–4.2) spore forming, Gram (-)ve (1.6–2.9), phosphate solubilizing (10–20), asymbiotic N₂-fixing (0.5–0.9), sulfur oxidizing (1.1–2.0), nitrifying (1.0–5.8) and denitrifying (12.1–18.7) bacteria; as well as, the actinomycetes (about 10⁴ c.f.u./g dr. soil) and fungi (about 10⁵ c.f.u./g dr. soil) were variable in the partially anaerobic, saline and drain out flooded rice soils during the post harvest period of the Himalayan, brackish water flooded, island and coastal habitats of India. The aerobic heterotrophic and spore forming bacteria were more than the anaerobic counterparts in the soils. Population (0.51–3.51 × 10⁶ c.f.u./g dr. soil) and crystal morphotype (spherical, bipyramidal and polymorphic) of the Bacillus thuringiensis (Bt) isolates of different soils were variable. Bt index was 0.002 at Mahe but 0.006 in other soils. The Bt isolates tolerated 5–12% NaCl. The osmolytes (mg/g dr. wt.) like the amino acids (0.38–99.45) and proline (0.38–0.80); and the antioxidative enzymes (units (U)/mg protein/min) viz. the catalase (0.17–5.59) and superoxide dismutase (0.35–74.46) were related with intrinsic osmotic stress tolerance of the Bt but they formed spores to overcome anoxic stress. Two Bt isolates were potent tolerant to both osmotic and anoxic stresses.     

Rapid solubilization of insoluble phosphate by a novel environmental stress-tolerant Burkholderia vietnamiensis M6 isolated from ginseng rhizospheric soil

We isolated and characterized novel insoluble phosphate (P)-solubilizing bacteria tolerant to environmental factors like high salt, low and high pHs, and low temperature. A bacterium M6 was isolated from a ginseng rhizospheric soil and confirmed to belong to Burkholderia vietnamiensis by BIOLOG system and 16S rRNA gene analysis. The optimal cultural conditions for the solubilization of P were 2.5% (w/v) glucose, 0.015% (w/v) urea, and 0.4% (w/v) MgCl₂·6H₂O along with initial pH 7.0 at 35°C. High-performance liquid chromatography analysis showed that B. vietnamiensis M6 produced gluconic and 2-ketogluconic acids. During the culture, the pH was reduced with increase in gluconic acid concentration and was inversely correlated with P solubilization. Insoluble P solubilization in the optimal medium was about 902 mg l⁻¹, which was approximately 1.6-fold higher than the yield in NBRIP medium (580 mg l⁻¹). B. vietnamiensis M6 showed resistance against different environmental stresses like 10–45°C, 1–5% (w/v) salt, and 2–11 pH range. The maximal concentration of soluble P produced by B. vietnamiensis M6 from Ca₃(PO₄)₂, CaHPO₄, and hydroxyapatite was 1,039, 2,132, and 1,754 mg l⁻¹, respectively. However, the strain M6 produced soluble P with 20 mg l⁻¹ from FePO₄ after 2 days and 100 mg l⁻¹ from AlPO₄ after 6 days, respectively. Our results indicate that B. vietnamiensis M6 could be a potential candidate for the development of biofertilizer applicable to environmentally stressed soil.     

Temperature-dependent phosphate solubilization by cold- and pH-tolerant species of Aspergillus isolated from Himalayan soil

Ten species of Aspergillus isolated from soil samples collected from different locations in the Indian Himalayan region have been studied for their growth requirements and tricalcium phosphate solubilization at different temperatures. The Aspergillus species could grow at low temperature and tolerated a wide range of pH. Phosphate solubilization by various Aspergillus species ranged between 374 μg/ml (A. candidus) to 1394 μg/ml (A. niger) at 28°C, 33 μg/ml (A. fumigatus) to 2354 μg/ml (A. niger) at 21°C, 93 μg/ml (A. fumigatus) to 1452 μg/ml (A. niger) at 14°C, and 21 μg/ml (A. wentii) to 83 μg/ml (A. niger) at 9°C. At 21 and 28°C, phosphate solubilization showed a decrease within 4 weeks of incubation, whereas at 9°C and 14°C, it continued further up to 6 weeks of incubation. In general, phosphate solubilization by different Aspergillus species was recorded at a maximum of 28°C or 21°C; biomass production was favored at 21°C or 14°C. Conversely, A. nidulans and A. sydowii exhibited maximum phosphate solubilization at 14°C and produced maximum biomass at 21°C. Data suggest that suboptimal conditions (higher or lower temperature) for fungal growth and biomass production were optimal for the production of metabolites involved in phosphate solubilization. Significant negative correlations were obtained between pH and phosphate solubilization for eight species at 28°C, for seven at 21°C, and for nine at 14°C. Extracellular phosphatase activity was exhibited only in case of A. niger, whreas intracellular phosphatase activity was detected in all species, the maximum being in A. niger. Statistically significant positive or negative correlations were obtained between phosphate solubilization and other parameters in most cases at different temperatures.     

Evaluation of pink-pigmented facultative methylotrophic bacteria for phosphate solubilization

Thirteen pink-pigmented facultative methylotrophic (PPFM) strains isolated from Adyar and Cooum rivers in Chennai and forest soil samples in Tamil Nadu, India, along with Methylobacterium extorquens, M. organophilum, M. gregans, and M. komagatae were screened for phosphate solubilization in plates. P-solubilization index of the PPFMs grown on NBRIP—BPB plates for 7 days ranged from 1.1 to 2.7. The growth of PPFMs in tricalcium phosphate amended media was found directly proportional to the glucose concentration. Higher phosphate solubilization was observed in four strains MSF 32 (415 mg l^−l), MDW 80 (301 mg l^−l), M. komagatae (279 mg l^−l), and MSF 34 (202 mg l^−l), after 7 days of incubation. A drop in the media pH from 6.6 to 3.4 was associated with an increase in titratable acidity. Acid phosphatase activity was more pronounced in the culture filtrate than alkaline phosphatase activity. Adherence of phosphate to densely grown bacterial surface was observed under scanning electron microscope after 7-day-old cultures. Biochemical characterization and screening for methanol dehydrogenase gene (mxaF) confirmed the strains as methylotrophs. The mxaF gene sequence from MSF 32 clustered towards M. lusitanum sp. with 99% similarity. This study forms the first detailed report on phosphate solubilization by the PPFMs.     

Ethyl Methanesulfonate Mutagenesis–Enhanced Mineral Phosphate Solubilization by Groundnut-Associated <Emphasis Type="Italic">Serratia marcescens</Emphasis> GPS-5

Twenty-three bacterial isolates were screened for their mineral phosphate–solubilizing (MPS) ability on Pikovskaya and National Botanical Research Institute’s phosphate (NBRIP) agar. The majority of the isolates exhibited a strong ability to solubilize hydroxyapatite in both solid and liquid media. The solubilization in liquid medium corresponded with a decrease in the pH of the medium. Serratia marcescens GPS-5, known for its biocontrol of late leaf spot in groundnut, emerged as the best solubilizer. S. marcescens GPS-5 was subjected to ethyl methanesulfonate (EMS) mutagenesis, and a total of 1700 mutants, resulting after 45 minutes of exposure, were screened on buffered NBRIP medium for alterations in MPS ability compared with that of the wild type. Seven mutants with increased (increased-MPS mutants) and 6 mutants with decreased (decreased-MPS mutants) MPS ability were isolated. All seven increased-MPS mutants were efficient at solubilizing phosphate in both solid and liquid NBRIP medium. Among the increased-MPS mutants, EMS XVIII Sm-35 showed the maximum (40%) increase in the amount of phosphate released in liquid medium compared with wild-type S. marcescens GPS-5, therefore, it would be a useful microbial inoculant in groundnut cultivation. EMS III Sm W, a nonpigmented mutant, showed the lowest solubilization of phosphate among the 6 decreased-MPS mutants.     

Evaluation of shelf life and rock phosphate solubilization of Burkholderia sp. in nutrient-amended clay, rice bran and rock phosphate-based granular formulation

Five phosphate-solubilizing bacteria (PSB) used in this study were isolated based on their ability to solubilize tricalcium phosphate (TCP) in Pikovskaya’s medium. Among the tested bacterial strains Burkholderia sp. strain CBPB-HIM showed the highest solubilization (363 μg of soluble P ml⁻¹) activity at 48 h of incubation. Further, this strain has been selected to assess its shelf life in nutrient-amended and -unamended clay, rice bran and rock phosphate (RP) pellet-based granular formulation. The results showed that the maximum viability of bacterium was observed in clay and rice bran (1:1) + 10% RP pellets than clay-RP pellets, irrespective of tested storage temperatures. Further, clay and rice bran (1:1) + 10% RP pellets amended with 1% glucose supported the higher number of cells compared to glycerol-amended and nutrient-unamended pellets. In this carrier solubilization of Morocco rock phosphate (MRP) by Burkholderia sp. strain CBPB-HIM was also investigated. The maximum of water and bicarbonate extractable P (206 and 245 μg P g⁻¹ of pellet respectively) was recorded in clay and rice bran (1:1) + 10% RP pellets amended with 1% glucose and glycerol respectively on day 5 of incubation. Therefore, this study proved the possibility of developing granular inoculant technology combining clay, rice bran and RP as substrates with phosphate-solubilizing Burkholderia.     

Biosolubilization of poorly soluble rock phosphates by Aspergillus tubingensis and Aspergillus niger

Three isolates of Aspergillus tubingensis and two isolates of Aspergillus niger isolated from rhizospheric soils were tested on solubilization of different rock phosphates. All the isolates of Aspergillus were capable of solubilizing all the natural rock phosphates. A. tubingensis (AT1) showed maximum percent solubilization in all the rock phosphates tested in this study when compared to other isolates. This isolate also showed highest phosphorus (P) solubilization when grown in the presence of 2% of rock phosphate. A. tubingensis (AT1) seems to be more efficient in solubilization of rock phosphates compared to other isolates reported elsewhere. This is the first report of rock phosphate solubilization by A. tubingensis and might provide an efficient large scale biosolubilization of rock phosphates intended for P fertilizer.     

Variation in the Nature of Organic Acid Secretion and Mineral Phosphate Solubilization by Citrobacter sp. DHRSS in the Presence of Different Sugars

A novel phosphate solubilizing bacterium (PSB) was isolated from the rhizosphere of sugarcane and is capable of utilizing sucrose and rock phosphate as the sole carbon and phosphate source, respectively. This PSB exhibited mineral phosphate solubilizing (MPS) phenotype on sugars such as sucrose and fructose, which are not substrates for enzyme glucose dehydrogenase (GDH), along with GDH substrates, viz., glucose, xylose, and maltose, as carbon sources. PCR amplification of the rRNA gene and sequence analysis identified this bacterium as Citrobacter sp. DHRSS. On sucrose and fructose Citrobacter sp. DHRSS liberated 170 and 100 μM free phosphate from rock phosphate and secreted 49 mM (2.94 g/L) and 35 mM (2.1 g/L) acetic acid, respectively. Growth of Citrobacter sp. DHRSS on sucrose is mediated by an intracellular inducible neutral invertase. Interestingly, in the presence of GDH substrates like glucose and maltose, Citrobacter sp. DHRSS produced approximately 20 mM (4.36 g/L) gluconic acid and phosphate released was 520 and 570 μM, respectively. Citrobacter sp. DHRSS GDH activity was found when grown on GDH and non-GDH substrates, indicating that it is constitutive and could act on a wide range of aldose sugars. This study demonstrates the role of different organic acids in mineral phosphate solubilization by rhizobacteria depending on the nature of the available carbon source.