Chemosphere 134 (2015) 1–6
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Phosphorus solubilization and plant growth enhancement by arsenic-resistant bacteria Piyasa Ghosh a,c, Bala Rathinasabapathi b, Lena Q. Ma a,c,⇑ a
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Jiangsu 210046, China Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, United States c Soil and Water Science Department, University of Florida, Gainesville, FL 32611, United States b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Arsenic-resistant bacteria were from
rhizosphere of arsenichyperaccumulator Pteris vittata. They produced siderophores and phytase to solubilize FePO4 and phytate and release P and Fe. They enhanced tomato Fe and P uptake and biomass accumulation.
a r t i c l e
i n f o
Article history: Received 5 February 2015 Received in revised form 21 March 2015 Accepted 23 March 2015
Handling Editor: J. de Boer Keywords: Arsenic-resistant bacteria FePO4 solubilization Phytate solubilization Tomato growth promotion
Bacterial siderophores
Plant growth enhancement
a b s t r a c t Phosphorus is an essential nutrient, which is limited in most soils. The P solubilization and growth enhancement ability of seven arsenic-resistant bacteria (ARB), which were isolated from arsenic hyperaccumulator Pteris vittata, was investigated. Siderophore-producing ARB (PG4, 5, 6, 9, 10, 12 and 16) were effective in solubilizing P from inorganic minerals FePO4 and phosphate rock, and organic phytate. To reduce bacterial P uptake we used filter-sterilized Hoagland medium containing siderophores or phytase produced by PG12 or PG6 to grow tomato plants supplied with FePO4 or phytate. To confirm that siderophores were responsible for P release, we compared the mutants of siderophore-producing bacterium Pseudomonas fluorescens Pf5 (PchA) impaired in siderophore production with the wild type and test strains. After 7 d of growth, mutant PchA solubilized 10-times less P than strain PG12, which increased tomato root biomass by 1.7 times. For phytate solubilization by PG6, tomato shoot biomass increased by 44% than control bacterium Pseudomonas chlororaphis. P solubilization by ARB from P. vittata may be useful in enhancing plant growth and nutrition in other crop plants. Published by Elsevier Ltd.
1. Introduction Phosphorus (P) is an essential nutrient required for optimum growth and development in living organisms including plants and bacteria (Huang et al., 2004). Although natural soil contains large amount of P (400–1200 mg kg 1), water-soluble P is only 1 mg kg 1 (Rodriguez and Fraga, 1999). Large reserve of insoluble ⇑ Corresponding author at: State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Jiangsu 210046, China. E-mail address: lqma@ufl.edu (L.Q. Ma). http://dx.doi.org/10.1016/j.chemosphere.2015.03.048 0045-6535/Published by Elsevier Ltd.
Solubilize FePO4 /phytate
P accumulates in soils from application of P fertilizers. In acidic soils, P is immobilized by Fe/Al minerals while in alkaline soils it is trapped by Ca/Mg carbonates. Organic P reserve in soil ranges from 5% to 95% of the total soil P, of which phytate (inositol phosphate) constitutes up to 50% (Rodriguez and Fraga, 1999). In soils, microbes can solubilize both inorganic and organic P by producing organic exudates such as organic acids, siderophore and phosphate solubilizing enzymes like phytase (Hayes et al., 2000; Li et al., 2006). It has been shown that plant growth promoting bacteria like Pseudomonas, Bacillus, and Burkholderia have the ability to solubilize P and make it more available to plants (Glick, 1995; Rodriguez and Fraga, 1999).
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Seven As-resistant bacteria (ARB) have been isolated from the rhizosphere of As-hyperaccumulator Pteris vittata from the field in North Central Florida (Ghosh et al., 2011). P. vittata prefers to grow in alkaline soils, which are characterized by low P and Fe availability and/or high concentrations of As (Lessl and Ma, 2013). Phosphorus and As compete for uptake into bacterial cells, so bacteria have developed an effective system to increase P solubility from soils. Bacterial siderophores effectively release As from insoluble minerals like FeAsO4 and AlAsO4 (Ghosh et al., 2011). In addition to inorganic P, a major source of organic P in plant rhizosphere is phytate so the studied bacteria may also produce phytase to extract P from phytate. Siderophores are low molecular weight organic ligands with high affinity and specificity for binding Fe. The stability constants of Fe3+-siderophore complexes range between 1023 to 1052 (Albrecht-Gary and Crumbliss, 1998; Ams et al., 2002). Bacterial ability to produce siderophores with high affinity for Fe3+ gives them selective advantage under Fe-limited conditions such as alkaline soils (Kraemer, 2004; Huang et al., 2004). Most siderophores bind strongly to trivalent ions like Fe3+ and Al3+, but their ability in binding divalent cations like Ca2+ is weak, with stability constant of only 102.64 (Kraemer, 2004). In this study we tested the ability of 7 ARB from the rhizosphere of P. vittata growing in As-rich soils in solubilizing P from insoluble minerals like FePO4 and phosphate rock [Ca10(PO4)6F2], and from organic P like phytate. These bacteria were selected because they were isolated from arsenic-rich soil with low Fe and P availability. The major objectives of this research were to assess the ability of siderophores and phytase produced by arsenic-resistant bacteria in: (1) solubilizing P from inorganic minerals like FePO4 and phosphate rock, and organic P like phytate, and (2) enhancing growth in tomato by increasing P and Fe uptake from FePO4 and phytate. 2. Materials and methods 2.1. Arsenic-resistant bacteria and control bacteria The 7 arsenic-resistant bacteria (ARB; PG4, 5, 6, 9, 10, 12 and 16) that were used for this study had different siderophore-producing ability ranging from 9.47 to 115 lM DFOM (deferroxamine mesylate) equiv./OD of cells (Ghosh et al., 2015). All bacteria were identified as Pseudomonas sp. by 16S rRNA sequencing. We also selected a control bacterium from the same genera so the results could be compared with the test strains. All bacteria were grown in modified LB media (pH 7) because the isolated bacteria were characterized in this media (Ghosh et al., 2015). We used 7 ARB for phosphate rock, FePO4 and phytate solubilization experiments and two bacteria (PG12 and PG6) with different siderophore and phytase producing ability for tomato growth experiment. For comparison, soil bacterium Pseudomonas chlororaphis 63-28 (PC), which was isolated from the rhizosphere of canola grown in non-contaminated soil (Paulitz et al., 2000), was also included. In addition to PC, we used wild type and two mutants of Pseudomonas fluorescens Pf-5 (PF) (PchA and PchC) to compare between siderophore-producing and non-producing mutants of the same bacteria. Mutant PchA is completely impaired in siderophore production whereas PchC is partially impaired (Hartney et al., 2011). 2.2. Bacterial solubilization of FePO4, phosphate rock, and phytate For the P solubilization experiments we used three insoluble P compounds: FePO4, phosphate rock, and phytate. Ground concentrate phosphate rock [Ca10(PO4)6F2] was obtained from White springs, Florida (Lessl and Ma, 2013). FePO4 as FePO4H2O and
phytate as phytic acid sodium salt hydrate from rice (C6H18Na12O24P6) were purchased from Sigma–Aldrich. Extracellularly-produced siderophores from 7 ARB and a control bacterium PC were collected from their spent growth media. Specifically, the bacteria were grown in modified LB medium for 24 h at 30 °C under 200 rpm shaking condition (Ghosh et al., 2011). Then the spent medium was filtered through 0.2 lM filter to remove bacteria. This filtered fraction of the spent media was again divided into two parts. One half was boiled for 5 min to inactivate enzyme activity and the other half was left intact. To test bacterial ability in solubilizing P, 1 mL of spent medium from the 7 ARB was incubated with 0.1 g of phosphate rock (PR), 0.01 g of FePO4 (FP) or 0.04 g phytate (PA) for 24 h at 30 °C under 200 rpm shaking condition. The amounts of insoluble P were used to ensure sufficient surface exposure with the spent growth medium for effective P solubilization. To test whether P was solubilized by siderophores via Fe chelation, we used 1 mL of the filter-sterilized spent medium of wild type and mutants PchA and PchC (Hartney et al., 2011) and incubated it with 0.01 g FePO4 for 24 h at 30 °C. The water soluble P was measured using modified molybdenum blue method (Ghosh et al., 2011).
2.3. Bacterial enhancement of tomato growth One-month old tomato seedlings were transferred to 0.2-strength Hoagland nutrient solution (HNS) at pH 7. The 0.2 HNS contained 6.2 mg L 1 P and 0.2 mg L 1 Fe. For the FePO4 experiment, the 0.2 HNS was provided with only 1 mg L 1 P and no Fe (low P Fe). For the phytate experiment, the 0.2 HNS was provided with 1 mg L 1 P and 0.2 mg L 1 Fe (low P + Fe). The tomato seedlings were acclimated in 0.2 HNS with (1) low P Fe or (2) low P + Fe for 35 d. The pH was checked regularly and the volume was maintained to 1 L in aerated containers during the experiment. For the FePO4 experiment, we selected two bacterial strains, PG12 (siderophore-producing ARB) and mutant PchA impaired in siderophore-production. The control was selected so that we can see the effect of siderophore in the bacterial spent media. For the phytate experiment, we selected two bacterial strains, PG6 (highest phytate solubilization among 7 ARB) and control strain P. chlororaphis (low phytate solubilization). Tomato plants acclimated in 0.2 HNS with low P and 0.2 mg L 1 Fe received 20 mL of spent medium from PG12 or PchA mutant and 0.2 g of phytate. The tomato plants acclimated in 0.2 HNS with low P and no Fe received 20 mL of spent medium from PG6 or PC and 0.2 g of FePO4. The plants were then grown for 1 week with 3 replicates. After 7-d of growth, plant biomass was determined after drying in oven at 80 °C for 2 d. To determine the role of P in enhancing plant growth, the dried plant samples were digested using H2SO4/H2O2 and was analyzed for P by modified molybdenum blue method (Ghosh et al., 2011). Total Fe concentration in the plant biomass was measured in the same extracts using inductively-coupled plasma emission spectroscopy (PerkinElmer 5300DV, Waltham, MA) via EPA Method 2007.
2.4. Statistical analysis All plant and bacterial experiments were conducted with three replicates for each treatment and every bacterial experiment was repeated twice. The analysis of variance (ANOVA) and Tukey’s mean grouping were used to determine significance difference between the treatment means. All statistical analyses were performed using SAS statistical software (SAS Inst., Cary, North Carolina, USA).
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3. Results and discussion 3.1. Bacterial solubilization of phosphate rock and FePO4 The spent medium was collected after growing bacteria for 24 h, which contained bacterial exudates without bacteria including organic acids, siderophore and phytase. Since those bacteria were isolated from an arsenic-rich soil with low Fe and P availability, it was expected that those bacteria have better ability to solubilize Fe and P compared to typical bacteria. To determine bacterial ability in solubilizing insoluble P mineral phosphate rock, we measured the P concentration in the spent media with and without addition of P mineral. The spent media without phosphate rock contained 0.08–0.74 mg L 1 P (data not shown). Among the 7 ARB tested, only 5 strains (PG4, 5, 9, 10, and 12) solubilized more P than the control bacterium PC (Table 1). PC was used a control because its P solubilization would be similar to other rhizosphere pseudomonad. The strains PG5 and PG12 were the most efficient in solubilizing P from phosphate rock (1.36–2.40 mg L 1) while PG16 and PG6 were unable to solubilize P (Table 1). To distinguish between enzymatic and siderophore-mediated P solubilization, the spent medium was boiled for 5 min to deactivate enzyme activity because siderophores are relatively tolerant to temperature (Kraemer, 2004). The 5 strains lost 58–84% of its P solubilization ability after boiling, indicating the importance of enzymes in P solubilization (Table 1). However, they all solubilized P after boiling, but it was less than unboiled extracts. The results indicated that both enzymatic and non-enzymatic processes contributed to P solubilization from phosphate rock by bacterial spent media. Similar results were also found in a study by Altomare et al. (1999) on a plant growth promoting fungi Trichoderma harzianum Rifai 1295-22. The cell-free extracts from the fungi solubilized 5 times more P from phosphate rock than the filtrate with fungal cells, which is attributed to chelating substances present in the spent media. The reduction in P solubilization in the filtrate with fungal cells may be due to the P uptake by live fungal cells after P solubilization. Based on previous studies, the primary mode of P solubilization by microbes is by production of organic acids (Vassilev et al., 2006; Chen et al., 2006), which is supported by pH reduction in the solution. Chen et al. (2006) also found an inverse relationship between the pH and P solubilization by bacterial strains Arthrobacter sp., Bacillus megaterium, and Serratia marcescens. The decrease in pH (6.8–7 to 4.9–6) was due to the exudation of organic acids like citric, gluconic, succinic and lactic acid. Another study by Vassilev et al. (2006) also proved that a fungal strain Aspergillus niger efficiently solubilized P (597 mg kg 1) from phosphate rock. In this case the pH was also lowered by the exudates produced by the fungus from 7 to 2.9. However, we did
not find significant pH change in our experiment, indicating that the solubilization of phosphate rock was probably not attributed to organic acid exudates. Overall, the amount of P solubilized from phosphate rock was limited partially due to the low chelation capacity of siderophores with Ca (Kraemer, 2004), so phosphate rock was not included in the further experiments. In addition to phosphate rock, we also determined the ability of bacterial spent media in solubilizing P from FePO4 (Table 1). Unlike phosphate rock, spent media from all 7 bacteria solubilized P from FePO4. Bacterial strain PG10 solubilized the least amount of P at 3.38 mg L 1 and PG12 the most P at 9.05 mg L 1 (Table 1). For example, PG12 solubilized 3.8-fold more P from FePO4 than phosphate rock (Table 1). We hypothesized that bacterial siderophores were partially responsible for P solubilization from FePO4. This hypothesis was supported by the strong correlation (r2 = 0.94) between bacterial siderophore-production and P solubilization from FePO4 (Table 1). After boiling, the spent media of PG4 did not lose its ability whereas other 6 strains lost 7.2–30% of their ability in P solubilization (Table 1). Though PG 5 and 12 were most efficient in P solubilization from both phosphate rock (65–84% enzymatic) and FePO4 (27–30% enzymatic), siderophore probably played a more important role in solubilizing FePO4 (Table 1). However, the data indicated that, besides siderophores, enzymatic process also played a role in P solubilization. Extracellular enzymes exuded by rhizobacteria have been found to improve plant growth and P uptake (Rodriguez et al., 2006). To further test our hypothesis that P was solubilized by bacterial siderophores, we tested wild type of P. fluorescens and two mutants PchA and PchC. While mutant PchA was completely impaired in siderophore production mutant PchC was partially impaired. The wild type (4.4 mg L 1) solubilized 2-fold and 4-fold more P than mutants PchC and PchA, which was consistent with their ability in siderophore production (Fig. 1). However, the fact that PchA solubilized 1.0 mg L 1 P indicated that other processes were also responsible for P solubilization from FePO4. Boiling reduced their ability of P solubilization by 22–33%, again implying limited enzyme-mediated P solubilization. The highest amount of enzymatic solubilization occurred in the filtered spent media of PG12 at 84% for phosphate rock and PG5 at 30% for FePO4 (Table 1). Apparently, different bacteria showed different enzymatic ability in solubilizing organic and inorganic P compounds. Fe-siderophore complex is more stable compared to that of Casiderophore (Albrecht-Gary and Crumbliss, 1998; Ams et al., 2002). For example, Fe3+-complex of trihydroxamate siderophore desferrioxamine-B (DFO-B) has a 1:1 stability constant of 1031 while that of Ca complex is only k = 102.6 (Martell et al., 2001). This was consistent with the lower P solubilized by bacterial spent media from
Table 1 Siderophore production and phosphate solubilized in 1 mL spent growth media by 7 arsenic-resistant bacteria from 0.01 g FePO4, 0.1 g phosphate rock and 0.04 g of phytate after 1 d of incubation. The values represent mean ± SE of three replicates. Phosphate compounds
Siderophore (lMol DFOM equiv./OD value of cells) Phosphate Enzymatic FePO4 Enzymatic Phytate Enzymatic Total Pb a b
rock (%) (%) (%)
Arsenic-resistant bacteria PC
PG16
PG10
PG9
PG6
PG4
PG5
PG12
18.6 ± 0.03 mg L 1 0.46 ± 0.03 43 3.30 ± 0.01 40 0.19 ± 0.01 11
15.8 ± 0.02
9.47 ± 0.02
10.8 ± 0.04
22.7 ± 0.01
NDa
73.2 ± 0.01
115 ± 0.02
00 – 3.90 ± 0.14 17 0.76 ± 0.03 80
1.05 ± 0.03 71 3.38 ± 0.12 12 0.53 ± 0.01 45
1.05 ± 0.15 77 4.12 ± 0.09 7.2 0.78 ± 0.06 58
00 – 4.88 ± 0.06 15 0.79 ± 0.04 47
0.51 ± 0.06 58 4.87 ± 0.36 0 0.77 ± 0.04 63
1.36 ± 0.03 65 5.77 ± 0.34 30 0.43 ± 0.06 28
2.41 ± 0.15 84 9.05 ± 0.36 27 0.41 ± 0.01 26
3.58 ± 0.03A
4.66 ± 0.2B
4.91 ± 0.2B
6.03 ± 0.3C
5.58 ± 0.16C
6.15 ± 0.46D
7.56 ± 0.43E
11.9 ± 0.56E
Not determined as the bacteria failed to grow in the CAS assay medium. Total = Sum of P solubilized by each bacteria from all three insoluble forms, phosphate rock, FePO4 and phytate.
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PO4 conc. (mgL-1)
5
A
demonstrated by their higher ability in solubilizing P from phosphate rock and FePO4 than the control bacterium PC.
SGM+FePO4
4
Boiled SGM+FePO4
a
3.2. Solubilization of phytate by bacteria spent media
3
B
b
2
C
c
1 0
WT
PchC
PchA
Fig. 1. P solubilization by two mutants of Pseudomonas fluorescens Pf-5 in 1 mL spent growth LB medium (SGM) before and after boiling. The bacteria were grown for 24 h then filtered through 0.2 lM membrane filter and added with 0.01 g of sterilized FePO4. The bars represent mean ± SE of three replicates. Treatments followed by different letters are not significantly different at a = 0.05.
phosphate rock than FePO4 (Table 1 and Fig. 2). The data also suggested that siderophore played a more important role than organic acids in solubilizing FePO4 than phosphate rock. Arsenate (AsV) is taken up into all living cells by phosphate transporters. Hence, AsV competes with P for entering into bacteria through P transporters. Arsenate forms insoluble compounds with Ca/Mg and Fe/Al (Silver and Phung, 2005) so it is mostly unavailable. Despite its well-established toxicity to life, microbes growing in As-rich environment are tolerant to arsenic. The presence of AsV in bacteria may induce P deficiency, making As-resistant bacteria more competitive in P solubilization from soil as well as in P uptake to survive under low P environment. This was
Biomass (g dw -1 /plant)
0.6
C
A
PC
B
0.4
PG6
A
0.3
c
0.2
ab
0.1 0
P concentration (mg g-1 dw)
Control (Low P+Fe)
0.5
10
Shoot
Root
The fact that boiling of spent growth media significantly reduced the ability of P solubilization from phosphate rock and FePO4 clearly indicated the importance of enzymatic processes in solubilizing P. Phytate accounts for 20–80% of the total organic P in soil (Richardson, 2001), so we tested the ability of spent bacterial media in solubilizing P from phytate. Similar to FePO4, spent media from all bacteria solubilized P from phytate, indicating their ability in producing phytase, the only enzyme known to release inorganic P from phytate (Table 1). However, our effort to determine phytase in the spent media was unsuccessful due to the interference of components in the media (data not shown). In addition, they were all more efficient than the control bacterium PC, with PG4, 6, 9 and 16 being more effective. PG12 and PG5, the most efficient in solubilizing P from phosphate rock and FePO4, were least efficient in solubilizing P from phytate (Table 1), whereas PG 6, the least effective in solubilizing P from phosphate rock (Table 1), was most effective in solubilizing P from phytate (0.89 mg L 1) (Table 1). The data clearly indicated that the 7 ARB had different ability in solubilizing phosphate rock, FePO4, and phytate via either enzymatic and/or non-enzymatic mechanisms. According to total P solubilized from the three P compounds, strains PG12 (11.9 mg L 1), PG5 (7.56 mg L 1) and PG4 (6.15 mg L 1) were more effective in extracting P (Table 1). Other strains PG9 (6.03 mg L 1), PG6 (5.58 mg L 1), and PG10 (4.91 mg L 1) showed lower ability of producing siderophores (9.50–22.7) also solubilized less P from insoluble P compounds (Table 1). Amongst the 7 bacteria, PG6 and PG16 failed to solubilize P from phosphate rock. The amount of P solubilized from phytate by spent bacterial media was more than that from phosphate rock but less than that from FePO4 (Table 1). Upon boiling, the amount of P solubilized from phytate was reduced by 26–80% (Table 1), indicating enzyme-controlled P solubilization. Among the bacteria, reduction due to boiling in PG16 was the highest (80%) whereas reduction in the control strain PC was the least at only 11%, which was consistent with their ability in P solubilization from phytate. The fact that after boiling still substantial amount of P was released from phytate indicated that boiling for 5 min did not completely deactivate bacterial phytase. Phytase is the only known enzyme to solubilize P from phytate (Lessl et al., 2013). It is a phosphomonoesterase enzyme, which aids in stepwise breakdown of phytate (Hariprasada and Niranjana, 2009). It is possible that temperature had limited effect on phytase.
c
B
B
b
3.3. Plant growth and nutrient uptake in tomato plants
8 6
A
A
a
4 2 0
Shoot
Root
Fig. 2. The effect of phytase-producing bacterium PG6 and control bacterium PC on plant growth (A) and P concentrations in tomato (B) after growing for 7 d in 0.2strength Hoagland solution with low P at 1 mg L 1 with and without 0.2 g of phytate. The bars represent mean ± SE of three replicates. Treatments followed by different letters are not significantly different at a = 0.05.
As PG6 was most effective in solubilizing P from phytate at 0.89 mg L 1 P (Table 1), spent media from PG6 was added to 0.2strength HNS containing low P at 1 mg L 1, with or without 0.2 g of phytate to grow tomato plants for 7 d. The addition of spent media from PG6 and control PC enhanced tomato growth than the control, with PG 6 being more effective than PC. The shoot biomass increased by 65% to 0.56 g per plant with PG6 addition compared to the control and 44% compared to control PC (Fig. 2A). We hypothesized that the increase in plant biomass was probably associated with the increased P uptake by tomato, which was supported by P data in tomato plant biomass (Fig. 2B). The P concentrations in tomato roots and shoots in PG6 treatment was 1.8–2.2 fold higher (8.63–9.20 mg g 1) than that in the control PC (4.1– 7.6 mg g 1).
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Since PG12 was the most effective in solubilizing P from FePO4, spent media from PG12 was added to 0.2-strength HNS containing low P (1 mg L 1) and no Fe, with and without 0.2 g of FePO4 to grow tomato for 7 d. The root and shoot biomass in PG12 treatment were 2.3–4.0 fold (0.04–0.07 g per plant) higher than that in the control and PchA mutant (Fig. 3A). The increased tomato biomass was probably associated with increased P and Fe uptake in the plant. The shoots and roots with PG12 treatment had 1.5–2.7 fold higher P concentration (7.46–11.6 mg g 1) than that in the control and 1.3–1.7 fold higher than that of the mutant PchA (Fig. 3B). The Fe concentrations in tomato plant also increased 10-fold (10.8 mg kg 1) in the shoots and 2-fold (270 mg kg 1) in the roots with PG12 treatment compared to PchA mutant (Fig. 3C). Previous studies on maize showed that seeds coated with P solubilizing bacteria increased grain yield by 64–85% compared to the seeds that were not coated due to P solubilization from phosphate rock by bacteria (Hameeda et al., 2008). Still the prospect of evaluating the role of siderophores in releasing insoluble FePO4 and phytase in solubilizing phytate in soil has not been explored extensively. Our study is the first of the few demonstrating the ability of the filtered spent media of arsenic-resistant bacteria in solubilizing phosphate rock, FePO4 and phytate and improving tomato growth.
Biomass (g dw-1/plant)
0.1
A B
0.08 0.06
c
A A
0.04
a
0.02 0
P concentration (mg g-1 dw)
Control (Low P-Fe) PchA PG12
Shoot
b
Root
14 12
C
B
10
b
B
8 6
a
a
A
4 2 0
Root
Fe concn (mg Kg -1 dw)
300 250 200
Shoot
C
C B A
150 100 50 0
b Root
a c Shoot
Fig. 3. Effect of siderophore-producing bacterium PG12 and mutant PchA (impaired in siderophore production) on plant growth (A), and concentrations of P (B) and Fe (C) in tomato biomass after growing for 7 d in 0.2-strength Hoagland solution with low P at 1 mg L 1 and no Fe with and without 0.2 g of FePO4. The bars represent mean ± SE of three replicates. Treatments followed by different letters are not significantly different at a = 0.05.
5
4. Conclusions We concluded that the spent growth media of arsenic-resistant bacteria were effective in solubilizing P from both organic (phytate) and inorganic (FePO4 and phosphate rock) via both enzymatic and/or non-enzymatic processes. We have also demonstrated that phytase-producing bacterial strain PG6 solubilized P from phytate and siderophore-producing PG12 solubilized P from FePO4, which significantly improved plant growth and nutrition in tomato seedlings. These bacterial strains may have a potential to be used in the field to improve plant P and Fe nutrition and thereby enhance plant growth. Acknowledgements This project is supported in part by UF-IFAS Innovation Grant. The authors thank Dr. Joyce Loper (Oregon State University) and Ed Davis (USDA-ARS HCRL, Corvallis, OR) for providing us with the wildtype and mutants of Pseudomonas fluorescens Pf-5. References Albrecht-Gary, A.M., Crumbliss, A.L., 1998. Coordination chemistry of siderophores: thermodynamics and kinetics of iron chelation and release. Met. Ions Biol. Syst. 35, 239–327. Altomare, C., Norvell, W.A., Björkman, T., Harman, G.E., 1999. Solubilization of phosphates and micronutrients by the plant-growth-promoting and biocontrol fungus Trichoderma harzianum Rifai 1295-22. Appl. Environ. Microbiol., 2926– 2933 Ams, D.A., Maurice, P.A., Hersman, L.E., Forsythe, J.H., 2002. Siderophore production by an aerobic Pseudomonas mendocina bacterium in the presence of kaolinite. Chem. Geol. 188, 161–170. Chen, Y.P., Rekha, P.D., Arun, A.B., Shen, F.T., Lai, W.-A., Young, C.C., 2006. Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities. Appl. Soil Ecol. 34, 33–41. Ghosh, P., Rathinasabapathi, B., Ma, L.Q., 2011. Arsenic-resistant bacteria solubilized arsenic in the growth media and increased growth of arsenic hyperaccumulator Pteris vittata L. Bioresour. Technol. 102, 8756–8761. Ghosh, P., Rathinasabapathi, B., Teplitski, M., Ma, L.Q., 2015. Bacterial ability in AsIII oxidation and AsV reduction: Relation to arsenic tolerance, P uptake, and siderophore production. Chemosphere. http://dx.doi.org/10.1016/ j.chemosphere.2014.12.046. Glick, B.R., 1995. The enhancement of plant growth by free living bacteria. Can. J. Microbiol. 42, 109–117. Hameeda, B., Harini, G., Rupela, O.P., Wani, S.P., Reddy, G., 2008. Growth promotion of maize by phosphate-solubilizing bacteria isolated from composts and macrofauna. Microbiol. Res. 163, 234–242. Hariprasada, P., Niranjana, S.R., 2009. Isolation and characterization of phosphate solubilizing rhizobacteria in improving plant health of tomato. Plant Soil 316, 13–24. Hartney, S.L., Mazurier, S., Kidarsa, T.A., Quccine, M.C., Lemanceau, P., Loper, J.E., 2011. TonB-dependent outer-membrane proteins and siderophore utilization in Pseudomonas fluorescens Pf-5. Biometals 24, 193–213. Hayes, J.E., Richardson, A.E., Simpson, R.J., 2000. Components of organic phosphorus in soil extracts that are hydrolysed by phytase and acid phosphatase. Biol. Fertil. Soils 32, 279–286. Huang, X., El-Alawi, Y., Penrose, D.M., Glick, B.R., Greenberg, B.M., 2004. Responses of three grass species to creosote during phytoremediation. Environ. Pollut. 130, 453–463. Kraemer, S.M., 2004. Iron oxide dissolution and solubility in the presence of siderophores. Aquat. Sci. 66, 3–18. Lessl, J.T., Ma, L.Q., 2013. Sparingly-soluble phosphate rock induced significant plant growth and arsenic uptake by Pteris vittata from three contaminated soils. Environ. Sci. Technol. 47, 5311–5318. Lessl, J.T., Ma, L.Q., Rathinasabapathi, B., Guy, C., 2013. Novel phytase from Pteris vittata resistant to arsenate, high temperature, and soil deactivation. Environ. Sci. Technol. 47, 2204–2211. Li, H., Smith, S.E., Holloway, R.E., Zhu, Y., Smith, F.A., 2006. Arbuscular mycorrhizal fungi contribute to P uptake by wheat grown in a phosphorus-fixing soil even in the absence of positive-growth responses. New Phytol. 172, 536–543. Martell, A.E., Smith, R.M., Motekaitis, R.J., 2001. NIST critically selected stability constants of metal complexes. NIST standard reference database 46, Version 6.0, NIST Gaithersburg. Paulitz, T., Nowak-Thompson, B., Gamard, P., Tsang, E., Loper, J., 2000. A novel antifungal furanone from Pseudomonas aureofaciens, a biocontrol agent of fungal plant pathogens. J. Chem. Ecol. 26, 1515–1524. Richardson, A.E., 2001. Prospects for using soil microorganism to improve the acquisition of phosphate by plant. Aust. J. Plant Physiol. 28, 897–906.
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Rodriguez, H., Fraga, R., 1999. Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnol. Adv. 17, 319–339. Rodriguez, H., Fraga, R., Gonzalez, T., Bashan, Y., 2006. Genetic of phosphate solubilization and its potential applications for improving plant growthpromoting bacteria. Plant Soil 287, 15–21.
Silver, S., Phung, L.T., 2005. Genes and enzymes involved in bacterial oxidation and reduction of inorganic arsenic. Appl. Environ. Microbiol. 71, 599–608. Vassilev, N., Medina, A., Azcon, R., Vassileva, M., 2006. Microbial solubilization of rock phosphate on media containing agro-industrial wastes and effect of the resulting products on plant growth and P uptake. Plant Soil 287, 77–84.
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Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Phosphorus solubilization and plant growth enhancement by arsenic-resistant bacteria Piyasa Ghosh a,c, Bala Rathinasabapathi b, Lena Q. Ma a,c,⇑ a
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Jiangsu 210046, China Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, United States c Soil and Water Science Department, University of Florida, Gainesville, FL 32611, United States b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Arsenic-resistant bacteria were from
rhizosphere of arsenichyperaccumulator Pteris vittata. They produced siderophores and phytase to solubilize FePO4 and phytate and release P and Fe. They enhanced tomato Fe and P uptake and biomass accumulation.
a r t i c l e
i n f o
Article history: Received 5 February 2015 Received in revised form 21 March 2015 Accepted 23 March 2015
Handling Editor: J. de Boer Keywords: Arsenic-resistant bacteria FePO4 solubilization Phytate solubilization Tomato growth promotion
Bacterial siderophores
Plant growth enhancement
a b s t r a c t Phosphorus is an essential nutrient, which is limited in most soils. The P solubilization and growth enhancement ability of seven arsenic-resistant bacteria (ARB), which were isolated from arsenic hyperaccumulator Pteris vittata, was investigated. Siderophore-producing ARB (PG4, 5, 6, 9, 10, 12 and 16) were effective in solubilizing P from inorganic minerals FePO4 and phosphate rock, and organic phytate. To reduce bacterial P uptake we used filter-sterilized Hoagland medium containing siderophores or phytase produced by PG12 or PG6 to grow tomato plants supplied with FePO4 or phytate. To confirm that siderophores were responsible for P release, we compared the mutants of siderophore-producing bacterium Pseudomonas fluorescens Pf5 (PchA) impaired in siderophore production with the wild type and test strains. After 7 d of growth, mutant PchA solubilized 10-times less P than strain PG12, which increased tomato root biomass by 1.7 times. For phytate solubilization by PG6, tomato shoot biomass increased by 44% than control bacterium Pseudomonas chlororaphis. P solubilization by ARB from P. vittata may be useful in enhancing plant growth and nutrition in other crop plants. Published by Elsevier Ltd.
1. Introduction Phosphorus (P) is an essential nutrient required for optimum growth and development in living organisms including plants and bacteria (Huang et al., 2004). Although natural soil contains large amount of P (400–1200 mg kg 1), water-soluble P is only 1 mg kg 1 (Rodriguez and Fraga, 1999). Large reserve of insoluble ⇑ Corresponding author at: State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Jiangsu 210046, China. E-mail address: lqma@ufl.edu (L.Q. Ma). http://dx.doi.org/10.1016/j.chemosphere.2015.03.048 0045-6535/Published by Elsevier Ltd.
Solubilize FePO4 /phytate
P accumulates in soils from application of P fertilizers. In acidic soils, P is immobilized by Fe/Al minerals while in alkaline soils it is trapped by Ca/Mg carbonates. Organic P reserve in soil ranges from 5% to 95% of the total soil P, of which phytate (inositol phosphate) constitutes up to 50% (Rodriguez and Fraga, 1999). In soils, microbes can solubilize both inorganic and organic P by producing organic exudates such as organic acids, siderophore and phosphate solubilizing enzymes like phytase (Hayes et al., 2000; Li et al., 2006). It has been shown that plant growth promoting bacteria like Pseudomonas, Bacillus, and Burkholderia have the ability to solubilize P and make it more available to plants (Glick, 1995; Rodriguez and Fraga, 1999).
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Seven As-resistant bacteria (ARB) have been isolated from the rhizosphere of As-hyperaccumulator Pteris vittata from the field in North Central Florida (Ghosh et al., 2011). P. vittata prefers to grow in alkaline soils, which are characterized by low P and Fe availability and/or high concentrations of As (Lessl and Ma, 2013). Phosphorus and As compete for uptake into bacterial cells, so bacteria have developed an effective system to increase P solubility from soils. Bacterial siderophores effectively release As from insoluble minerals like FeAsO4 and AlAsO4 (Ghosh et al., 2011). In addition to inorganic P, a major source of organic P in plant rhizosphere is phytate so the studied bacteria may also produce phytase to extract P from phytate. Siderophores are low molecular weight organic ligands with high affinity and specificity for binding Fe. The stability constants of Fe3+-siderophore complexes range between 1023 to 1052 (Albrecht-Gary and Crumbliss, 1998; Ams et al., 2002). Bacterial ability to produce siderophores with high affinity for Fe3+ gives them selective advantage under Fe-limited conditions such as alkaline soils (Kraemer, 2004; Huang et al., 2004). Most siderophores bind strongly to trivalent ions like Fe3+ and Al3+, but their ability in binding divalent cations like Ca2+ is weak, with stability constant of only 102.64 (Kraemer, 2004). In this study we tested the ability of 7 ARB from the rhizosphere of P. vittata growing in As-rich soils in solubilizing P from insoluble minerals like FePO4 and phosphate rock [Ca10(PO4)6F2], and from organic P like phytate. These bacteria were selected because they were isolated from arsenic-rich soil with low Fe and P availability. The major objectives of this research were to assess the ability of siderophores and phytase produced by arsenic-resistant bacteria in: (1) solubilizing P from inorganic minerals like FePO4 and phosphate rock, and organic P like phytate, and (2) enhancing growth in tomato by increasing P and Fe uptake from FePO4 and phytate. 2. Materials and methods 2.1. Arsenic-resistant bacteria and control bacteria The 7 arsenic-resistant bacteria (ARB; PG4, 5, 6, 9, 10, 12 and 16) that were used for this study had different siderophore-producing ability ranging from 9.47 to 115 lM DFOM (deferroxamine mesylate) equiv./OD of cells (Ghosh et al., 2015). All bacteria were identified as Pseudomonas sp. by 16S rRNA sequencing. We also selected a control bacterium from the same genera so the results could be compared with the test strains. All bacteria were grown in modified LB media (pH 7) because the isolated bacteria were characterized in this media (Ghosh et al., 2015). We used 7 ARB for phosphate rock, FePO4 and phytate solubilization experiments and two bacteria (PG12 and PG6) with different siderophore and phytase producing ability for tomato growth experiment. For comparison, soil bacterium Pseudomonas chlororaphis 63-28 (PC), which was isolated from the rhizosphere of canola grown in non-contaminated soil (Paulitz et al., 2000), was also included. In addition to PC, we used wild type and two mutants of Pseudomonas fluorescens Pf-5 (PF) (PchA and PchC) to compare between siderophore-producing and non-producing mutants of the same bacteria. Mutant PchA is completely impaired in siderophore production whereas PchC is partially impaired (Hartney et al., 2011). 2.2. Bacterial solubilization of FePO4, phosphate rock, and phytate For the P solubilization experiments we used three insoluble P compounds: FePO4, phosphate rock, and phytate. Ground concentrate phosphate rock [Ca10(PO4)6F2] was obtained from White springs, Florida (Lessl and Ma, 2013). FePO4 as FePO4H2O and
phytate as phytic acid sodium salt hydrate from rice (C6H18Na12O24P6) were purchased from Sigma–Aldrich. Extracellularly-produced siderophores from 7 ARB and a control bacterium PC were collected from their spent growth media. Specifically, the bacteria were grown in modified LB medium for 24 h at 30 °C under 200 rpm shaking condition (Ghosh et al., 2011). Then the spent medium was filtered through 0.2 lM filter to remove bacteria. This filtered fraction of the spent media was again divided into two parts. One half was boiled for 5 min to inactivate enzyme activity and the other half was left intact. To test bacterial ability in solubilizing P, 1 mL of spent medium from the 7 ARB was incubated with 0.1 g of phosphate rock (PR), 0.01 g of FePO4 (FP) or 0.04 g phytate (PA) for 24 h at 30 °C under 200 rpm shaking condition. The amounts of insoluble P were used to ensure sufficient surface exposure with the spent growth medium for effective P solubilization. To test whether P was solubilized by siderophores via Fe chelation, we used 1 mL of the filter-sterilized spent medium of wild type and mutants PchA and PchC (Hartney et al., 2011) and incubated it with 0.01 g FePO4 for 24 h at 30 °C. The water soluble P was measured using modified molybdenum blue method (Ghosh et al., 2011).
2.3. Bacterial enhancement of tomato growth One-month old tomato seedlings were transferred to 0.2-strength Hoagland nutrient solution (HNS) at pH 7. The 0.2 HNS contained 6.2 mg L 1 P and 0.2 mg L 1 Fe. For the FePO4 experiment, the 0.2 HNS was provided with only 1 mg L 1 P and no Fe (low P Fe). For the phytate experiment, the 0.2 HNS was provided with 1 mg L 1 P and 0.2 mg L 1 Fe (low P + Fe). The tomato seedlings were acclimated in 0.2 HNS with (1) low P Fe or (2) low P + Fe for 35 d. The pH was checked regularly and the volume was maintained to 1 L in aerated containers during the experiment. For the FePO4 experiment, we selected two bacterial strains, PG12 (siderophore-producing ARB) and mutant PchA impaired in siderophore-production. The control was selected so that we can see the effect of siderophore in the bacterial spent media. For the phytate experiment, we selected two bacterial strains, PG6 (highest phytate solubilization among 7 ARB) and control strain P. chlororaphis (low phytate solubilization). Tomato plants acclimated in 0.2 HNS with low P and 0.2 mg L 1 Fe received 20 mL of spent medium from PG12 or PchA mutant and 0.2 g of phytate. The tomato plants acclimated in 0.2 HNS with low P and no Fe received 20 mL of spent medium from PG6 or PC and 0.2 g of FePO4. The plants were then grown for 1 week with 3 replicates. After 7-d of growth, plant biomass was determined after drying in oven at 80 °C for 2 d. To determine the role of P in enhancing plant growth, the dried plant samples were digested using H2SO4/H2O2 and was analyzed for P by modified molybdenum blue method (Ghosh et al., 2011). Total Fe concentration in the plant biomass was measured in the same extracts using inductively-coupled plasma emission spectroscopy (PerkinElmer 5300DV, Waltham, MA) via EPA Method 2007.
2.4. Statistical analysis All plant and bacterial experiments were conducted with three replicates for each treatment and every bacterial experiment was repeated twice. The analysis of variance (ANOVA) and Tukey’s mean grouping were used to determine significance difference between the treatment means. All statistical analyses were performed using SAS statistical software (SAS Inst., Cary, North Carolina, USA).
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3. Results and discussion 3.1. Bacterial solubilization of phosphate rock and FePO4 The spent medium was collected after growing bacteria for 24 h, which contained bacterial exudates without bacteria including organic acids, siderophore and phytase. Since those bacteria were isolated from an arsenic-rich soil with low Fe and P availability, it was expected that those bacteria have better ability to solubilize Fe and P compared to typical bacteria. To determine bacterial ability in solubilizing insoluble P mineral phosphate rock, we measured the P concentration in the spent media with and without addition of P mineral. The spent media without phosphate rock contained 0.08–0.74 mg L 1 P (data not shown). Among the 7 ARB tested, only 5 strains (PG4, 5, 9, 10, and 12) solubilized more P than the control bacterium PC (Table 1). PC was used a control because its P solubilization would be similar to other rhizosphere pseudomonad. The strains PG5 and PG12 were the most efficient in solubilizing P from phosphate rock (1.36–2.40 mg L 1) while PG16 and PG6 were unable to solubilize P (Table 1). To distinguish between enzymatic and siderophore-mediated P solubilization, the spent medium was boiled for 5 min to deactivate enzyme activity because siderophores are relatively tolerant to temperature (Kraemer, 2004). The 5 strains lost 58–84% of its P solubilization ability after boiling, indicating the importance of enzymes in P solubilization (Table 1). However, they all solubilized P after boiling, but it was less than unboiled extracts. The results indicated that both enzymatic and non-enzymatic processes contributed to P solubilization from phosphate rock by bacterial spent media. Similar results were also found in a study by Altomare et al. (1999) on a plant growth promoting fungi Trichoderma harzianum Rifai 1295-22. The cell-free extracts from the fungi solubilized 5 times more P from phosphate rock than the filtrate with fungal cells, which is attributed to chelating substances present in the spent media. The reduction in P solubilization in the filtrate with fungal cells may be due to the P uptake by live fungal cells after P solubilization. Based on previous studies, the primary mode of P solubilization by microbes is by production of organic acids (Vassilev et al., 2006; Chen et al., 2006), which is supported by pH reduction in the solution. Chen et al. (2006) also found an inverse relationship between the pH and P solubilization by bacterial strains Arthrobacter sp., Bacillus megaterium, and Serratia marcescens. The decrease in pH (6.8–7 to 4.9–6) was due to the exudation of organic acids like citric, gluconic, succinic and lactic acid. Another study by Vassilev et al. (2006) also proved that a fungal strain Aspergillus niger efficiently solubilized P (597 mg kg 1) from phosphate rock. In this case the pH was also lowered by the exudates produced by the fungus from 7 to 2.9. However, we did
not find significant pH change in our experiment, indicating that the solubilization of phosphate rock was probably not attributed to organic acid exudates. Overall, the amount of P solubilized from phosphate rock was limited partially due to the low chelation capacity of siderophores with Ca (Kraemer, 2004), so phosphate rock was not included in the further experiments. In addition to phosphate rock, we also determined the ability of bacterial spent media in solubilizing P from FePO4 (Table 1). Unlike phosphate rock, spent media from all 7 bacteria solubilized P from FePO4. Bacterial strain PG10 solubilized the least amount of P at 3.38 mg L 1 and PG12 the most P at 9.05 mg L 1 (Table 1). For example, PG12 solubilized 3.8-fold more P from FePO4 than phosphate rock (Table 1). We hypothesized that bacterial siderophores were partially responsible for P solubilization from FePO4. This hypothesis was supported by the strong correlation (r2 = 0.94) between bacterial siderophore-production and P solubilization from FePO4 (Table 1). After boiling, the spent media of PG4 did not lose its ability whereas other 6 strains lost 7.2–30% of their ability in P solubilization (Table 1). Though PG 5 and 12 were most efficient in P solubilization from both phosphate rock (65–84% enzymatic) and FePO4 (27–30% enzymatic), siderophore probably played a more important role in solubilizing FePO4 (Table 1). However, the data indicated that, besides siderophores, enzymatic process also played a role in P solubilization. Extracellular enzymes exuded by rhizobacteria have been found to improve plant growth and P uptake (Rodriguez et al., 2006). To further test our hypothesis that P was solubilized by bacterial siderophores, we tested wild type of P. fluorescens and two mutants PchA and PchC. While mutant PchA was completely impaired in siderophore production mutant PchC was partially impaired. The wild type (4.4 mg L 1) solubilized 2-fold and 4-fold more P than mutants PchC and PchA, which was consistent with their ability in siderophore production (Fig. 1). However, the fact that PchA solubilized 1.0 mg L 1 P indicated that other processes were also responsible for P solubilization from FePO4. Boiling reduced their ability of P solubilization by 22–33%, again implying limited enzyme-mediated P solubilization. The highest amount of enzymatic solubilization occurred in the filtered spent media of PG12 at 84% for phosphate rock and PG5 at 30% for FePO4 (Table 1). Apparently, different bacteria showed different enzymatic ability in solubilizing organic and inorganic P compounds. Fe-siderophore complex is more stable compared to that of Casiderophore (Albrecht-Gary and Crumbliss, 1998; Ams et al., 2002). For example, Fe3+-complex of trihydroxamate siderophore desferrioxamine-B (DFO-B) has a 1:1 stability constant of 1031 while that of Ca complex is only k = 102.6 (Martell et al., 2001). This was consistent with the lower P solubilized by bacterial spent media from
Table 1 Siderophore production and phosphate solubilized in 1 mL spent growth media by 7 arsenic-resistant bacteria from 0.01 g FePO4, 0.1 g phosphate rock and 0.04 g of phytate after 1 d of incubation. The values represent mean ± SE of three replicates. Phosphate compounds
Siderophore (lMol DFOM equiv./OD value of cells) Phosphate Enzymatic FePO4 Enzymatic Phytate Enzymatic Total Pb a b
rock (%) (%) (%)
Arsenic-resistant bacteria PC
PG16
PG10
PG9
PG6
PG4
PG5
PG12
18.6 ± 0.03 mg L 1 0.46 ± 0.03 43 3.30 ± 0.01 40 0.19 ± 0.01 11
15.8 ± 0.02
9.47 ± 0.02
10.8 ± 0.04
22.7 ± 0.01
NDa
73.2 ± 0.01
115 ± 0.02
00 – 3.90 ± 0.14 17 0.76 ± 0.03 80
1.05 ± 0.03 71 3.38 ± 0.12 12 0.53 ± 0.01 45
1.05 ± 0.15 77 4.12 ± 0.09 7.2 0.78 ± 0.06 58
00 – 4.88 ± 0.06 15 0.79 ± 0.04 47
0.51 ± 0.06 58 4.87 ± 0.36 0 0.77 ± 0.04 63
1.36 ± 0.03 65 5.77 ± 0.34 30 0.43 ± 0.06 28
2.41 ± 0.15 84 9.05 ± 0.36 27 0.41 ± 0.01 26
3.58 ± 0.03A
4.66 ± 0.2B
4.91 ± 0.2B
6.03 ± 0.3C
5.58 ± 0.16C
6.15 ± 0.46D
7.56 ± 0.43E
11.9 ± 0.56E
Not determined as the bacteria failed to grow in the CAS assay medium. Total = Sum of P solubilized by each bacteria from all three insoluble forms, phosphate rock, FePO4 and phytate.
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PO4 conc. (mgL-1)
5
A
demonstrated by their higher ability in solubilizing P from phosphate rock and FePO4 than the control bacterium PC.
SGM+FePO4
4
Boiled SGM+FePO4
a
3.2. Solubilization of phytate by bacteria spent media
3
B
b
2
C
c
1 0
WT
PchC
PchA
Fig. 1. P solubilization by two mutants of Pseudomonas fluorescens Pf-5 in 1 mL spent growth LB medium (SGM) before and after boiling. The bacteria were grown for 24 h then filtered through 0.2 lM membrane filter and added with 0.01 g of sterilized FePO4. The bars represent mean ± SE of three replicates. Treatments followed by different letters are not significantly different at a = 0.05.
phosphate rock than FePO4 (Table 1 and Fig. 2). The data also suggested that siderophore played a more important role than organic acids in solubilizing FePO4 than phosphate rock. Arsenate (AsV) is taken up into all living cells by phosphate transporters. Hence, AsV competes with P for entering into bacteria through P transporters. Arsenate forms insoluble compounds with Ca/Mg and Fe/Al (Silver and Phung, 2005) so it is mostly unavailable. Despite its well-established toxicity to life, microbes growing in As-rich environment are tolerant to arsenic. The presence of AsV in bacteria may induce P deficiency, making As-resistant bacteria more competitive in P solubilization from soil as well as in P uptake to survive under low P environment. This was
Biomass (g dw -1 /plant)
0.6
C
A
PC
B
0.4
PG6
A
0.3
c
0.2
ab
0.1 0
P concentration (mg g-1 dw)
Control (Low P+Fe)
0.5
10
Shoot
Root
The fact that boiling of spent growth media significantly reduced the ability of P solubilization from phosphate rock and FePO4 clearly indicated the importance of enzymatic processes in solubilizing P. Phytate accounts for 20–80% of the total organic P in soil (Richardson, 2001), so we tested the ability of spent bacterial media in solubilizing P from phytate. Similar to FePO4, spent media from all bacteria solubilized P from phytate, indicating their ability in producing phytase, the only enzyme known to release inorganic P from phytate (Table 1). However, our effort to determine phytase in the spent media was unsuccessful due to the interference of components in the media (data not shown). In addition, they were all more efficient than the control bacterium PC, with PG4, 6, 9 and 16 being more effective. PG12 and PG5, the most efficient in solubilizing P from phosphate rock and FePO4, were least efficient in solubilizing P from phytate (Table 1), whereas PG 6, the least effective in solubilizing P from phosphate rock (Table 1), was most effective in solubilizing P from phytate (0.89 mg L 1) (Table 1). The data clearly indicated that the 7 ARB had different ability in solubilizing phosphate rock, FePO4, and phytate via either enzymatic and/or non-enzymatic mechanisms. According to total P solubilized from the three P compounds, strains PG12 (11.9 mg L 1), PG5 (7.56 mg L 1) and PG4 (6.15 mg L 1) were more effective in extracting P (Table 1). Other strains PG9 (6.03 mg L 1), PG6 (5.58 mg L 1), and PG10 (4.91 mg L 1) showed lower ability of producing siderophores (9.50–22.7) also solubilized less P from insoluble P compounds (Table 1). Amongst the 7 bacteria, PG6 and PG16 failed to solubilize P from phosphate rock. The amount of P solubilized from phytate by spent bacterial media was more than that from phosphate rock but less than that from FePO4 (Table 1). Upon boiling, the amount of P solubilized from phytate was reduced by 26–80% (Table 1), indicating enzyme-controlled P solubilization. Among the bacteria, reduction due to boiling in PG16 was the highest (80%) whereas reduction in the control strain PC was the least at only 11%, which was consistent with their ability in P solubilization from phytate. The fact that after boiling still substantial amount of P was released from phytate indicated that boiling for 5 min did not completely deactivate bacterial phytase. Phytase is the only known enzyme to solubilize P from phytate (Lessl et al., 2013). It is a phosphomonoesterase enzyme, which aids in stepwise breakdown of phytate (Hariprasada and Niranjana, 2009). It is possible that temperature had limited effect on phytase.
c
B
B
b
3.3. Plant growth and nutrient uptake in tomato plants
8 6
A
A
a
4 2 0
Shoot
Root
Fig. 2. The effect of phytase-producing bacterium PG6 and control bacterium PC on plant growth (A) and P concentrations in tomato (B) after growing for 7 d in 0.2strength Hoagland solution with low P at 1 mg L 1 with and without 0.2 g of phytate. The bars represent mean ± SE of three replicates. Treatments followed by different letters are not significantly different at a = 0.05.
As PG6 was most effective in solubilizing P from phytate at 0.89 mg L 1 P (Table 1), spent media from PG6 was added to 0.2strength HNS containing low P at 1 mg L 1, with or without 0.2 g of phytate to grow tomato plants for 7 d. The addition of spent media from PG6 and control PC enhanced tomato growth than the control, with PG 6 being more effective than PC. The shoot biomass increased by 65% to 0.56 g per plant with PG6 addition compared to the control and 44% compared to control PC (Fig. 2A). We hypothesized that the increase in plant biomass was probably associated with the increased P uptake by tomato, which was supported by P data in tomato plant biomass (Fig. 2B). The P concentrations in tomato roots and shoots in PG6 treatment was 1.8–2.2 fold higher (8.63–9.20 mg g 1) than that in the control PC (4.1– 7.6 mg g 1).
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Since PG12 was the most effective in solubilizing P from FePO4, spent media from PG12 was added to 0.2-strength HNS containing low P (1 mg L 1) and no Fe, with and without 0.2 g of FePO4 to grow tomato for 7 d. The root and shoot biomass in PG12 treatment were 2.3–4.0 fold (0.04–0.07 g per plant) higher than that in the control and PchA mutant (Fig. 3A). The increased tomato biomass was probably associated with increased P and Fe uptake in the plant. The shoots and roots with PG12 treatment had 1.5–2.7 fold higher P concentration (7.46–11.6 mg g 1) than that in the control and 1.3–1.7 fold higher than that of the mutant PchA (Fig. 3B). The Fe concentrations in tomato plant also increased 10-fold (10.8 mg kg 1) in the shoots and 2-fold (270 mg kg 1) in the roots with PG12 treatment compared to PchA mutant (Fig. 3C). Previous studies on maize showed that seeds coated with P solubilizing bacteria increased grain yield by 64–85% compared to the seeds that were not coated due to P solubilization from phosphate rock by bacteria (Hameeda et al., 2008). Still the prospect of evaluating the role of siderophores in releasing insoluble FePO4 and phytase in solubilizing phytate in soil has not been explored extensively. Our study is the first of the few demonstrating the ability of the filtered spent media of arsenic-resistant bacteria in solubilizing phosphate rock, FePO4 and phytate and improving tomato growth.
Biomass (g dw-1/plant)
0.1
A B
0.08 0.06
c
A A
0.04
a
0.02 0
P concentration (mg g-1 dw)
Control (Low P-Fe) PchA PG12
Shoot
b
Root
14 12
C
B
10
b
B
8 6
a
a
A
4 2 0
Root
Fe concn (mg Kg -1 dw)
300 250 200
Shoot
C
C B A
150 100 50 0
b Root
a c Shoot
Fig. 3. Effect of siderophore-producing bacterium PG12 and mutant PchA (impaired in siderophore production) on plant growth (A), and concentrations of P (B) and Fe (C) in tomato biomass after growing for 7 d in 0.2-strength Hoagland solution with low P at 1 mg L 1 and no Fe with and without 0.2 g of FePO4. The bars represent mean ± SE of three replicates. Treatments followed by different letters are not significantly different at a = 0.05.
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4. Conclusions We concluded that the spent growth media of arsenic-resistant bacteria were effective in solubilizing P from both organic (phytate) and inorganic (FePO4 and phosphate rock) via both enzymatic and/or non-enzymatic processes. We have also demonstrated that phytase-producing bacterial strain PG6 solubilized P from phytate and siderophore-producing PG12 solubilized P from FePO4, which significantly improved plant growth and nutrition in tomato seedlings. These bacterial strains may have a potential to be used in the field to improve plant P and Fe nutrition and thereby enhance plant growth. Acknowledgements This project is supported in part by UF-IFAS Innovation Grant. The authors thank Dr. Joyce Loper (Oregon State University) and Ed Davis (USDA-ARS HCRL, Corvallis, OR) for providing us with the wildtype and mutants of Pseudomonas fluorescens Pf-5. References Albrecht-Gary, A.M., Crumbliss, A.L., 1998. Coordination chemistry of siderophores: thermodynamics and kinetics of iron chelation and release. Met. Ions Biol. Syst. 35, 239–327. Altomare, C., Norvell, W.A., Björkman, T., Harman, G.E., 1999. Solubilization of phosphates and micronutrients by the plant-growth-promoting and biocontrol fungus Trichoderma harzianum Rifai 1295-22. Appl. Environ. Microbiol., 2926– 2933 Ams, D.A., Maurice, P.A., Hersman, L.E., Forsythe, J.H., 2002. Siderophore production by an aerobic Pseudomonas mendocina bacterium in the presence of kaolinite. Chem. Geol. 188, 161–170. Chen, Y.P., Rekha, P.D., Arun, A.B., Shen, F.T., Lai, W.-A., Young, C.C., 2006. Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities. Appl. Soil Ecol. 34, 33–41. Ghosh, P., Rathinasabapathi, B., Ma, L.Q., 2011. Arsenic-resistant bacteria solubilized arsenic in the growth media and increased growth of arsenic hyperaccumulator Pteris vittata L. Bioresour. Technol. 102, 8756–8761. Ghosh, P., Rathinasabapathi, B., Teplitski, M., Ma, L.Q., 2015. Bacterial ability in AsIII oxidation and AsV reduction: Relation to arsenic tolerance, P uptake, and siderophore production. Chemosphere. http://dx.doi.org/10.1016/ j.chemosphere.2014.12.046. Glick, B.R., 1995. The enhancement of plant growth by free living bacteria. Can. J. Microbiol. 42, 109–117. Hameeda, B., Harini, G., Rupela, O.P., Wani, S.P., Reddy, G., 2008. Growth promotion of maize by phosphate-solubilizing bacteria isolated from composts and macrofauna. Microbiol. Res. 163, 234–242. Hariprasada, P., Niranjana, S.R., 2009. Isolation and characterization of phosphate solubilizing rhizobacteria in improving plant health of tomato. Plant Soil 316, 13–24. Hartney, S.L., Mazurier, S., Kidarsa, T.A., Quccine, M.C., Lemanceau, P., Loper, J.E., 2011. TonB-dependent outer-membrane proteins and siderophore utilization in Pseudomonas fluorescens Pf-5. Biometals 24, 193–213. Hayes, J.E., Richardson, A.E., Simpson, R.J., 2000. Components of organic phosphorus in soil extracts that are hydrolysed by phytase and acid phosphatase. Biol. Fertil. Soils 32, 279–286. Huang, X., El-Alawi, Y., Penrose, D.M., Glick, B.R., Greenberg, B.M., 2004. Responses of three grass species to creosote during phytoremediation. Environ. Pollut. 130, 453–463. Kraemer, S.M., 2004. Iron oxide dissolution and solubility in the presence of siderophores. Aquat. Sci. 66, 3–18. Lessl, J.T., Ma, L.Q., 2013. Sparingly-soluble phosphate rock induced significant plant growth and arsenic uptake by Pteris vittata from three contaminated soils. Environ. Sci. Technol. 47, 5311–5318. Lessl, J.T., Ma, L.Q., Rathinasabapathi, B., Guy, C., 2013. Novel phytase from Pteris vittata resistant to arsenate, high temperature, and soil deactivation. Environ. Sci. Technol. 47, 2204–2211. Li, H., Smith, S.E., Holloway, R.E., Zhu, Y., Smith, F.A., 2006. Arbuscular mycorrhizal fungi contribute to P uptake by wheat grown in a phosphorus-fixing soil even in the absence of positive-growth responses. New Phytol. 172, 536–543. Martell, A.E., Smith, R.M., Motekaitis, R.J., 2001. NIST critically selected stability constants of metal complexes. NIST standard reference database 46, Version 6.0, NIST Gaithersburg. Paulitz, T., Nowak-Thompson, B., Gamard, P., Tsang, E., Loper, J., 2000. A novel antifungal furanone from Pseudomonas aureofaciens, a biocontrol agent of fungal plant pathogens. J. Chem. Ecol. 26, 1515–1524. Richardson, A.E., 2001. Prospects for using soil microorganism to improve the acquisition of phosphate by plant. Aust. J. Plant Physiol. 28, 897–906.
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