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Phosphate-solubilising bacteria enhance Oryza sativa growth and nutrient accumulation in an oxisol fertilized with rock phosphate
Ecological Engineering 83 (2015) 380–385
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Phosphate-solubilising bacteria enhance Oryza sativa growth and nutrient accumulation in an oxisol fertilized with rock phosphate Elaine Martins da Costa, Wellington de Lima, Silvia M. Oliveira-Longatti, Fatima M. de Souza* Setor de Biologia, Microbiologia e Processos Biológicos do Solo, Departamento de Ciência do Solo, Universidade Federal de Lavras, Campus UFLA, 37200-000 Lavras, Minas Gerais, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 February 2015 Received in revised form 10 June 2015 Accepted 28 June 2015 Available online xxx
This study aimed to evaluate the ability of phosphate-solubilizing bacteria (PSB) to enhance growth and nutrient accumulation of rice plants grown in an Oxisol fertilized with Bayóvar rock phosphate (BRP). The experiment was entirely randomised and consisted of the following 12 treatments: individual inoculation with five PSB strains in pots containing BRP; a control with BRP without inoculation; five different soluble phosphorus (P) concentrations (50, 100, 150, 200 and 250 mg P2O5 dm 3) applied as NH4H2PO4 and a control without P or inoculation. The PSB strains UFLA 04-21 (Burkholderia sp.), UFLA 0310 (Paenibacillus kribbensis), UFPI B5-6 (Enterobacter sp.) and UFPI B5-8A (Pseudomonas sp.) increased the shoot dry matter, root dry matter and total dry matter, the number of tillers and the accumulation of phosphorus, nitrogen, calcium, magnesium, sulphur and boron compared with the treatment with BRP without inoculation. The increases in the dry matter of the shoots and roots were 52% and 120%, respectively, with the strain UFLA 03-10 (Paenibacillus kribbensis) inoculation treatment. The four strains promoted shoot dry matter that were equivalent to approximately 60% of the shoot dry matter produced in the treatment with 150 mg dm 3 of soluble P and increased the P accumulation in the shoots compared with the treatments with 50 and 100 mg P2O5 dm 3 of soluble P. These results indicate that inoculation with PSB combined with rock phosphate is an economical and sustainable strategy for improving the growth and nutrient accumulation of the rice plants. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Plant growth promoting rhizobacteria Biofertilizers Bayóvar rock phosphate
1. Introduction Phosphorus (P) is one of the most limiting nutrients for crop development in tropical soils because of its complex dynamics. In addition to the low natural content available for uptake by plants, most of the P applied to the soil is retained in its particles because of the adsorption and precipitation reactions that are favoured by the acidity and oxidic mineralogy of most of these soils producing inorganic phosphates (Novais and Smyth, 1999). Thus, high doses of phosphate fertilisers are required to obtain satisfactory yields. Because of the potential risk of depleting natural reserves of soluble phosphates (SPs), the high cost and the residual effect of SPs when applied in large amounts, the use of alternative P sources, such as less concentrated rock phosphates (RPs) that are lower in cost, has been suggested (Fageria et al., 1991; Korndorfer et al., 1999; Léon et al., 1986). However, RPs dissolve more slowly than SPs, and the dissolution varies between the different types of
* Corresponding author: Fax: +55 35 3829 12 51. http://dx.doi.org/10.1016/j.ecoleng.2015.06.045 0925-8574/ ã 2015 Elsevier B.V. All rights reserved.
commercially available RPs. Typically, igneous and metamorphic RPs, such as Brazilian RPs, demonstrate low reactivity, while sedimentary phosphates are more reactive and, therefore, have been used more frequently in agriculture (Dias et al., 2014; Léon et al., 1986; Resende et al., 2006). The more reactive RPs include Bayóvar rock phosphate (BRP), which is of sedimentary origin and is formed by the deposition and subsequent decomposition of marine animal remains from the Piura region (province of Sechura) in Peru (Léon et al., 1986). This phosphate containing approximately 28% P2O5 and 31% calcium, is insoluble in water and is approximately 13% soluble in citric acid. In Brazil, few studies on BRP have been conducted, but it has been shown that this phosphate can partially replace the use of SPs on agricultural crops, indicating its potential use in agriculture (Dias et al., 2014). An important method for increasing the utilisation efficiency of RPs and/or reducing the use of SPs fertilisers is the use of bacteria that solubilise inorganic phosphates and could potentially increase the amount of P available to plants (Estrada et al., 2013; Lavakush et al., 2014; Oliveira-Longatti et al., 2013, 2014; Pereira and Castro, 2014; Shankar et al,. 2011). These bacteria participate in the
E. Martins da Costa et al. / Ecological Engineering 83 (2015) 380–385
solubilisation process through various mechanisms. The mechanism most often cited in the literature is the production and release of low molecular weight organic acids. However organic acid production does not always correlate with the solubilisation of inorganic phosphates (Marra et al., 2012). Other reported mechanisms include extrusion of protons via cellular respiration and the uptake of ammonium as a nitrogen source (Illmer and Schinner, 1992), exopolysaccharide production (Yi et al., 2008) and siderophore production (Hamdali et al., 2008). In Brazil, rice (Oryza sativa L.) is one of the main cereals grown, with an estimated planted area of approximately 2427,000 hectares and an estimated production of 12 million tons for the years 2011–2012 (CONAB, 2013). Rice grown under rainfed systems, despite occupying approximately two-thirds of the total cultivated area, accounts for approximately one-third of the national yield (Crusciol et al., 2005). This low production is partially due to the fact that most of this rice is grown in Cerrado soils, which tend to have low P availability in most cases. Therefore, to achieve high productivity, the crop requires high doses of this nutrient, which results in higher production costs. The combined use of low cost RPs and inoculation with phosphate-solubilising bacteria (PSB) is an alternative technique to improve the nutrition, growth and yield of rice and to partially offset the cost of SP fertilisers. Furthermore, some PSB also contribute to the growth and nutrition of crops by participating in other biological processes (Costa et al., 2013; Oliveira-Longagatti et al., 2013, 2014). In general, few studies investigating the combined application of PSB and RPs have been reported, and there are no reports of this type for rice crop grown in Brazilian soils. Thus, the aim of this study was to evaluate the growth and nutrient accumulation of rice plants inoculated with inorganic phosphatesolubilising bacteria and grown in pots containing Oxisol fertilized with Bayóvar rock phosphate. 2. Materials and methods 2.1. Strains evaluated Five strains of bacteria belonging to the collection of the Laboratory of Soil Microbiology, UFLA (Universidade Federal de Lavras—Federal University of Lavras) were used in this study. The origin, accession numbers of 16S rRNA gene sequence and plant growth promoting characteristics of these strains are shown in Table 1.
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2.2. Count of calcium phosphate-solubilising bacteria in the soil Prior to the experiment, a single soil sample was collected from each pot to form a composite sample and to estimate the number of calcium phosphate-solubilising bacteria in the soil. Serial dilutions of the soil sample up to 10 7 were prepared in saline (8,5 g L 1 NaCl). Streaking with Drigalski spatula was performed using 100 mL of the dilution suspensions on plates containing NBRIP culture medium (Nautiyal, 1999) which were incubated at 28 C. Counts were periodically conducted until the eighth day after inoculation. 2.3. Experiment in greenhouse The experiment was conducted in a greenhouse from December 2013 to March 2014. Polypropylene pots with a 4 dm3 capacity that contained yellow–red Latosol (Oxisol) (EMBRAPA, 2006) with a clayey texture and a low P content, collected from the 0–20 cm layer in Lavras, Minas Gerais. Table 2 lists the chemical and physical characteristics of the soil before liming and fertilisation. Based on the chemical analysis of the soil, liming was performed to increase the base saturation to 50% using calcium carbonate (CaCO3) and magnesium carbonate (MgCO3) at a 4:1 ratio. The soil was incubated for 40 days with a moisture of approximately 60% of the total pore volume. The experimental design was completely randomised with four replicates. The following 12 treatments were used in this study: individual inoculation with five PSB strains (UFLA 04-21, UFLA 04155, UFLA 03-10, UFPI B5-6 and UFPI B5-8A) in pots with soil containing Bayóvar RP (150 mg P2O5 dm 3); a control with Bayóvar RP (150 mg P2O5 dm 3) without inoculation; five different concentrations of soluble phosphate (50, 100, 150, 200 and 250 mg P2O5 dm 3) applied as monoammonium phosphate (MAP) (NH4H2PO4) and a control without P or inoculation. Fertilisation was conducted as follows for all treatments (mg dm 3): 450 of N, 350 of K and 40 of S, provided as Urea [(NH2)2CO], Potassium chloride (KCl) and potassium sulphate (K2SO4), respectively, and 1.5 of Cu, 3.6 of Mn, 5.0 of Zn, 0.8 of B and 0.15 of Mo provided as copper sulphate (CuSO45H2O), manganese chloride (MnCl24H2O), zinc sulphate (ZnSO47H2O), boric acid (H3BO3) and ammonium molybdate [(NH4)6Mo7O244H2O], respectively. The calculations to determine the N dose took into consideration the amounts already provided by NH4H2PO4 at the various P concentrations. The nitrogen and potassium fertilisation treatments were divided into five applications, the first occurred at
Table 1 Origin and plant growth promoting characteristics of the strains evaluated. Strains
Location/ LUS
Phylogenetic affiliation
Accession No. in GenBank of 16S rRNA sequences (NCBI)
Plant growth promoting characteristics(c,d,e) IAA in DYGS Free-living nitrogen medium fixation (mg ml 1)* Tryptophan
In vitro solubilization
CaHPO4 AlH6P3O12 FePO4.2H2O – UFLA 0421 UFLA 04155 UFLA 0310 UFPI B5-6 UFPI B58A
+
AM/AG
Burkholderia sp..(a)
FJ534643
+
+
ND
2.53 2.14
+
AM/SFI
Burkholderia fungorum (b) Paenibacillus kribbensis (c) Enterobacter sp.(d) Pseudomonas sp.(d)
GU144370
+
+
ND
6.29 4.53
+
JQ041885
+
–
+
ND
ND
KJ879611 KJ879613
+ +
– –
– –
2.87 26.54 – 5.40 9.71 –
MG/STF PI/AG PI/AG
ND
LUS—Land-use systems; AM—Amazônia state; MG—Minas Gerais estate; PI—Piauí estate; AG—Agriculture; SFI—Secondary forest in an initial stage of regeneration; STF— Semideciduous tropical forest; ND—Not determined; *Production of indole-3-acetic acid (IAA) in DYGS medium without ( ) and with (+) tryptophan. (a)Lima et al., 2009; (b) Silva et al., 2012; (c)Marra et al., 2012; (d)Costa et al. unpublished data; (e)Oliveira-Longatti et al., 2013.
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Table 2 Chemical and physical characteristics of soil sample before implantation of the experiment. Depth cm
pH H2O
Pa mg dm
K+
Ca2+ cmolc dm
0–20
5.4
0.84
Depth cm
O.M %
P-rem ml L 1
Zn mg dm
0–20
2.11
3.54
1.59
3
24.00
Mg2+
Al3+
H+Al
TB
ECE
T
V %
m
0.10
0.10
2.90
1.86
1.96
4.76
39.11
5.10
3
1.70 Fe
Mn
Cu
B
S
Sand %
Silt
Clay
29.78
4.98
3.15
0.13
28.07
17
10
73
3
a
Mehlich-1 method; TB—Total base cations; ECE—Effective cation exchange capacity; T—Potential cation exchange capacity; V—index of base saturation; m—index of aluminium saturation; O.M—Organic matter.
moistened filter paper and cotton, where they remained for 72 h in an incubator at 28 C until the emergence of rootlets. Six seeds were sown per pot. Eight days after emergence, thinning to two seedlings was performed. The bacterial strains were cultured in liquid medium 79 (Fred and Waksman, 1928) under stirring of 110 rpm at 28 C for 48 h. For each inoculated treatment, 1 mL of
planting followed by applications 30, 45, 60 and 75 days after planting. Before planting, rice seeds (cultivar BRS-MG Relâmpago, recommended for rainfed farming systems) were surface sterilised using 98% ethanol (30 s), 2% sodium hypochlorite (two minutes) and subsequent successive washes in sterile distilled water. Next, the seeds were placed in sterilised Petri dishes containing
70
a
16
a b
TDM SDM TN
c
g pot
-1
c d
10 d c
c d
d
20
e e
10
d
a
d
8 a
b
d
d d
e
e
b
c
d
30
12
b
c
-1
50
40
14
a a
d
c
6
c
Tillers number pot
60
4 e 2
f f
200
150
250 Solu ble P
c
ble P
c
Solu
d
P 10 0
d
Solu ble P
d
Solu ble
d
Solu ble P
hout nd w it out I a
e
b b a
UFLA
30
e
150 mg
P
g pot
-1
20
W ith
10
50
0 f
BRP 04-1 55 + 150 BRP UFLA 03-1 0+1 50 B RP UFLA 04-2 1+1 50 B RP UF P I B58A + 150 BRP UFP I B56+1 50 B RP
0
40
50
RDM
60
250 ble P Solu
200
P 15 0
P 10 0
Solu ble P
Solu ble
Solu ble
P 50 Solu ble
BRP 04-1 55 + 150 BRP UFLA 03-1 0+1 50 B RP UFLA 04-2 1+1 50 B RP UF P I B58A + 150 BRP UF P I B56+1 50 B RP
150 mg
UFLA
W ith
out I a
nd w it
hout
P
70
Fig. 1. Total dry matter (TDM), shoot dry matter (SDM), tillers number (TN) and root dry matter (RDM) of rice plants (Oryza sativa L.) grown under different conditions of phosphorus supply for 90 days after planting in pots with a Oxisol. Means followed by the same letter do not differ by the Scott–Knott test at a 5% probability. BRP: Bayóvar rock phosphate (mg P2O5 dm 3) without inoculation; soluble P = Monoammonium phosphate (mg P2O5 dm 3); UFLA 04-155 (Burkholderia fungorum); UFLA 04-21 (Burkholderia sp.); UFLA 03-10 (Paenibacillus kribbensis); UFPI B5-6 (Enterobacter sp.); UFPI B5-8A (Pseudomonas sp.).
E. Martins da Costa et al. / Ecological Engineering 83 (2015) 380–385
inoculum at a concentration of 1 108 bacterial cells mL added for each seed sown.
1
was
2.4. Plant sampling and analysis Plants were harvested 90 days after planting, and the number of tillers per plant (NT) was assessed. Then, the shoots were washed with distilled water. The plant material was dried in a convection oven at 60 C to determine the shoot dry matter (SDM), root dry matter (RDM), total dry matter (TDM) and the accumulation of macronutrients [nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and sulphur (S)] and micronutrients [copper (Cu), iron (Fe), manganese (Mn), zinc (Zn) and Boron (B)] in the shoots (Malavolta et al., 1997). 2.5. pH and phosphorus available in the rhizospheric soil after the experiment After collecting the plants, rhizospheric soil samples were taken from the pots of each treatment group to determine the pH (H2O) and the available P extracted using the Mehlich-1 method (Mehlich, 1953). 2.6. Statistical analysis According to the Shapiro–Wilks test, the test data were normally distributed and were subjected to analysis of variance (ANOVA), using the statistical analysis program Sisvar 5.3 (Ferreira, 2011). The effects of the treatments were compared by the Scott– Knott test at P < 0.05. 3. Results 3.1. Count of calcium phosphate-solubilising bacteria in the soil Calcium phosphate-solubilising bacteria were detected in the studied soil up to a dilution of 10 3. The estimated number of these organisms was 2.1 104 CFU g 1 soil. 3.2. Influence of PSB inoculation on growth and nutrient accumulation in rice plants The treatments had a significant effect on the production of SDM, RDM and TDM, on the NT (Fig. 1) and on the accumulation of macro- and micro-nutrients (Table 3). Four (UFLA 04-21, UFLA 03-
383
10, UFPI B5-6 and UFPI B5-8A) of the five strains promoted the production of SDM, RDM and TDM and NT to similar levels that were greater than those observed for the treatment with Bayóvar RP without inoculation (Fig. 1). The SDM increased to 42% and 52% in the treatments inoculated with UFLA 04-21 (Burkholderia sp.) and UFLA 03-10 (Paenibacillus kribbensis), respectively, compared with the treatment with Bayóvar RP without inoculation. Greater increases of 65%, 105%, 111% and 120% in the RDM were observed in the treatments inoculated with UFPI B5-8A (Pseudomonas sp.), UFLA 04-21 (Burkholderia sp.), UFPI B5-6 (Enterobacter sp.) and UFLA 03-10 (Paenibacillus kribbensis), respectively. However, compared with the treatments with SP, these strains produced lower yields of SDM, RDM and TDM; they only produced more tillers than the treatment with 50 mg P2O5 dm 3 (Fig. 1). The strain UFLA 04-155 yielded results that were similar to those of the treatment with BRP without inoculation for all the growth parameters assessed. Comparison of the treatments with different concentrations of SP revealed that applying 200 mg P2O5 dm 3 is sufficient for promoting maximal rice plant growth (Fig. 1). In this treatment, the SDM production and NT results were similar to the results from the treatment with the highest SP concentration (250 mg P2O5 dm 3), and the production of RDM and TDM were greater than that of the treatment with the highest SP concentration. Regarding the accumulation of P in shoots, the treatments inoculated with the strains UFLA 04-21, UFLA 03-10, UFPI B5-6 and UFPI B5-8A yielded significantly higher accumulations of P than the treatment with Bayóvar RP without inoculation and the treatments with 50 and 100 mg P2O5 dm 3 SP (Table 3). The increase in P accumulation in the shoot was 13% and 39% for the treatments inoculated with the strains UFPI B5-8A and UFLA 03-10, respectively, compared with the treatment with Bayóvar RP without inoculation. Most notably, the strain UFLA 03-10 yielded a P accumulation in the shoots that was similar to P accumulation resulting from the treatments with 200 and 250 mg P2O5 dm 3 SP. Significant increases in the accumulation of the other macronutrients in the shoots (N, K, Ca, Mg and S) were also observed for the treatments inoculated with the strains UFLA 04-21, UFLA 0310, UFPI B5-6 and UFPI B5-8A, with the exception of K accumulation for the treatment inoculated with the strains UFPI B5-6 and UFPI B5-8A, which were similar to the treatment with BRP without inoculation (Table 3). All of the treatments with BRP yielded significant increases in Ca accumulation compared with the treatments with SP. Most notably, the inoculation treatments with the strains UFLA 03-10, UFPI B5-8A and UFLA 04-21 yielded
Table 3 Accumulation of macronutrients and micronutrients in shoots of rice plants (Oryza sativa L.) grown under different conditions of phosphorus supply for 90 days after planting in pots with a Oxisol. Treatments Without I Without P 150 mg P2O5 dm 3 (BRP) UFLA 04-155 + 150 mg P2O5 dm 3(BRP) UFLA 04-21 + 150 mg P2O5 dm 3(BRP) UFLA 03-10 + 150 mg P2O5 dm 3(BRP) UFPI B5-6 + 150 mg P2O5 dm 3 BRP) UFPI B5-8A + 150 mg P2O5dm 3 (BRP) 50 mg P2O5dm 3 (MAP) 100 mg P2O5 dm 3 (MAP) 150 mg P2O5 dm 3 (MAP) 200 mg P2O5 dm 3 (MAP) 250 mg P2O5 dm 3 (MAP) CV (%)
N (mg pot 8.88 f 326.05 e 302.10 e 431.97 d 490.38 c 440.55 d 405.30 d 404.28 d 520.62 c 631.48 b 766.58 a 758.85 a 7.22
P 1
K
Ca
Mg
S
) 0.09 e 27.16 c 25.09 c 34.21 b 37.73 a 34.68 b 30.59 b 19.02 d 26.35 c 31.39 b 40.88 a 36.57 a 11.08
3.15 c 244.32 b 221.27 b 350.90 a 405.19 a 286.51 b 249.15 b 389.45 a 449.07 a 421.66 a 469.15 a 391.93 a 16.56
0.74 f 60.93 c 55.08 c 86.05 a 92.74 a 73.16 b 87.60 a 13.60 e 31.95 d 39.28 d 34.60 d 40.98 d 10.30
1.58 f 31.78 e 28.56 e 50.74 d 47.57 d 45.21 d 43.50 d 46.39 d 59.27 c 66.82 c 83.17 a 73.63 b 12.08
0.82 e 46.45 d 40.24 d 60.64 c 61.65 c 63.28 c 59.04 c 53.67 c 70.93 b 75.51 b 82.84 a 73.78 b 10.58
Cu (mg pot 2.50 e 173.90 d 152.33 d 211.99 c 209.03 c 225.20 b 183.45 d 231.56 b 234.90 b 272.14 a 296.13 a 270.34 a 8.94
1
Fe
Mn
Zn
B
742.5 f 1035.68 e 1422.38 d 1506.36 d 1405.33 d 1193.18 e 1336.92 d 1682.71 d 2333.88 c 2371.87 c 3439.03 b 3873.08 a 12.06
113.50 c 8616.05 b 7959.65 b 9614.68 a 8756.58 b 8391.23 b 9194.36 a 9583.97a 9559.88 a 9803.06 a 9175.58 a 8555.49 b 6.97
17.00 f 2554.89 d 1701.77 e 3880.50 c 3244.02 d 3727.41 c 3097.00 d 4115.73 c 5433.35 b 5734.74 b 6722.34 a 5956.12 b 9.84
5.75 e 194.72 d 168.91 d 318.92 b 248.79 c 283.86 b 370.41 a 379.93 a 366.68 a 397.93 a 378.44 a 386.20 a 10.28
)
BRP—Bayóvar rock phosphate; MAP—Monoammonium phosphate; UFLA 04-155 (Burkholderia fungorum); UFLA 04-21 (Burkholderia sp.); UFLA 03-10 (Paenibacillus kribbensis); UFPI B5-6 (Enterobacter sp.); UFPI B5-8A (Pseudomonas sp.). Values in the same column with different letters are significantly different by the Scott–Knott test (P < 0.05).
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participate in other processes that promote plant growth (Table 1). However, we emphasise that it is not always possible to correlate in vitro activity with in vivo effects on plant growth and nutrition. For example, despite its ability to solubilise insoluble inorganic phosphates in vitro, the strain UFLA 04-155 (Burkholderia fungorum) did not appear to promote the growth of rice plants in the present study. Of the macro- and micro-nutrients assessed, this strain only increased the accumulation of Fe. Strains belonging to the genera Burkholderia, Enterobacter, Paenibacillus and Pseudomonas are often described as potential promoters of plant growth because of their ability to participate in various biological processes. In rice crops, significant increases in the growth of shoots and roots were observed in plants inoculated with strains of Burkholderia (Estrada et al., 2013; Souza et al., 2013), Enterobacter (Habibi et al., 2014; Shankar et al., 2011), Paenibacillus (Bal et al., 2013; Beneduzi et al., 2008) and Pseudomonas (Habibi et al., 2014; Lavakush et al., 2014; Yasmin et al., 2004). However, only two of these previous studies (Estrada et al., 2013; Lavakush et al., 2014) evaluated the contribution of phosphate solubilisation to the growth and nutrition of rice in pots with non-sterile soil. Estrada et al., (2013) demonstrated that inoculation with a Burkholderia vietnamiensis strain increased SDM production, P and N accumulation and the yield of rice grown in pots with soil fertilised with tricalcium phosphate [Ca3(PO4)2]. The combination of the Pseudomonas, Azotobacter chroococcum and Azospirillum brasilense strains and 30 kg ha 1 P2O5 (unnamed source) in an experiment conducted in pots that contained soil resulted in greater plant growth, greater accumulation of N, P and K and a greater yield of rice compared with the treatment without inoculation with 30 kg ha 1 P2O5 (Lavakush et al., 2014). The ability of strains belonging to the genera Enterobacter and Paenibacillus to solubilise calcium phosphate has been reported in several previous studies (Beneduzi et al., 2008; Costa et al., 2013; Krey et al., 2013; Marra et al., 2012; Shankar et al., 2011). The contribution of calcium phosphate solubilisation to growth and plant nutrition has also been reported in grasses, such as maize inoculated with strains belonging to the Enterobacter genus (Krey et al., 2013). However, our study is the first to report the contribution of strains belonging to the genera Enterobacter and Paenibacillus to the growth and nutrition of rice crops through the solubilisation of inorganic rock phosphate. According to the literature, lowering the pH of the solution by releasing the organic acids in the rhizosphere is one of the main mechanisms responsible for the solubilisation of insoluble inorganic phosphates in soils (Lin et al., 2006). However, the soil solution pH did not change after the experiment for any of the treatments in the present study. This result is consistent with those of Marra et al., (2011), who demonstrated that the solubilisation of
52%, 44% and 41% greater Ca accumulation, respectively, compared with the treatment with BRP without inoculation. The accumulation of micro-nutrients in the shoots varied depending on the treatment. Inoculation with the strain UFLA 04155 only yielded a greater accumulation of Fe, while inoculation with the strain UFLA 04-21 resulted in a greater accumulation of all the micro-nutrients assessed (Cu, Fe, Mn, Zn and B) compared with the treatment with Bayóvar RP without inoculation (Table 3). The strain UFLA 03-10 yielded a greater accumulation of Cu, Fe and B. The strain UFPI B5-6 increased the accumulation of Cu, Zn and B. The strain UFPI B5-8A increased the accumulation of Fe, Mn and B. The treatment with UFLA 04-155 inoculation exhibited results that were similar to the results for the treatment with only Bayóvar RP in terms of all of the macro-nutrients and most of the micronutrients (Cu, Mn and B) accumulated (Table 3). 3.3. pH (H2O) and available phosphorus in the rhizospheric soil after the experiment The pH (H2O) values of the soil did not vary significantly among the various treatments after the experiment. However, the treatments significantly affected the level of P available in the rhizospheric soil (Table 4). The treatment with Bayóvar RP without inoculation resulted in a significantly higher level of available P (12.93 mg dm 3) than the other treatments. The treatments including inoculation with the UFLA 04-21 and UFPI B5-8A strains yielded similar values (11.54 and 11.08 mg of P dm 3) that were greater than those for the remaining strains (Table 4). 4. Discussion The combined use of low-cost RPs and inoculation with PSB is an important strategy for reducing the use of SP fertilisers and production costs while increasing the sustainability of agroecosystems. Of the five bacterial strains evaluated in this study, four contributed to the growth and nutrition of rice plants by solubilising the inorganic calcium phosphate with soil containing Bayóvar RP. Strains belonging to the genera Burkholderia (UFLA 0421), Enterobacter (UFPI B5-6), Paenibacillus (UFLA 03-10) and Pseudomonas (UFPI B5-8A) were able to significantly increase the production of SDM, RDM and TDM, the NT and the accumulation of P, N, Ca, Mg, S and B compared to the treatment with Bayóvar RP without inoculation. Although the studied soil contained a native population of calcium phosphate-solubilising bacteria of approximately 2.1 104 CFU g 1 of soil, the four studied strains mentioned were more efficient. In previous studies, these strains have demonstrated the ability to solubilise insoluble inorganic phosphates in vitro and to
Table 4 pH (H2O) of the rhizosphere soil samples and available phosphorus (P) from each treatment after the conducting experiment. Treatments
pH (H2O)
Available P (Mehlich-1) (mg dm
Without I Without P 150 mg P2O5 dm 3 (BRP) UFLA 04-155 + 150 mg P2O5 dm 3 (BRP) UFLA 04-21 + 150 mg P2O5 dm 3 (BRP) UFLA 03-10 + 150 mg P2O5 dm 3 (BRP) UFPI B5-6 + 150 mg P2O5 dm 3 (BRP) UFPI B5-8A + 150 mg P2O5 dm 3 (BRP) 50 mg P2O5 dm 3 (MAP) 100 mg P2O5 dm 3 (MAP) 150 mg P2O5 dm 3 (MAP) 200 mg P2O5 dm 3 (MAP) 250 mg P2O5 dm 3 (MAP) CV (%)
4.7 a 4.9 a 4.8 a 4.8 a 4.9 a 4.9 a 5.1 a 4.8 a 4.9 a 4.9 a 4.8 a 4.9 a 1.91
0.63 f 12.93 a 7.39 c 11.54 b 7.76 c 5.14 d 11.02 b 0.56 f 1.27 f 1.71 f 1.56 f 3.20 e 15.78
3
)
RP—Bayóvar rock phosphate; MAP—Monoammonium phosphate; UFLA 04-155 (Burkholderia fungorum); UFLA 04-21 (Burkholderia sp.); UFLA 03-10 (Paenibacillus kribbensis); UFPI B5-6 (Enterobacter sp.); UFPI B5-8A (Pseudomonas sp.). Values in the same column with different letters are significantly different by the Scott–Knott test (P < 0.05).
E. Martins da Costa et al. / Ecological Engineering 83 (2015) 380–385
insoluble inorganic phosphates is not always associated with the lowering of pH. We found the highest levels of available P in the soil solutions treated with BRP and the lowest levels in the soil solutions treated with SP. This finding may be due to the extraction method used (Mehlich, 1953), which despite its standard use for assessing the availability of P in soils fertilised with soluble sources, may be inappropriate for soils fertilised with RPs because this method begins with the extraction process of solubilisation by H+ ions, possibly promoting the excessive dissolution of particles of RPs and thus overestimating the levels of available P (Doll et al., 1960; Cabala and Wild, 1982). In our study, the soluble P levels resulting from the treatments with BRP were most likely overestimated because there was no observed relationship between the levels in the soil and the amount of P that accumulated in the shoots. Considering the potential risk of depleting more naturally concentrated phosphate reserves and the high cost of SP fertilisers, it is important to use alternate methods, such as those used in our study, that at least partially replace SPs. In this case, the combined use of BRP and inoculation with bacterial strains that efficiently solubilise insoluble phosphates was shown to be a good strategy. The strains that contributed to the growth and nutrition of rice plants in the present study will be evaluated under field conditions in future studies to verify their potential as crop inoculants. 5. Conclusions Inoculation of rice with strains UFLA 04-21 (Burkholderia sp.), UFPI B5-6 (Enterobacter sp.), UFLA 03-10 (Paenibacillus kribbensis) and UFPI B5-8A (Pseudomonas sp.) enhanced plant growth and nutrient accumulation by solubilization of insoluble calcium phosphate. These four strains may be used as biofertilizers, constituting an economical and sustainable strategy to increase rice yield. Acknowledgments The authors thank the National Council for Scientific and Technological Development [Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)], the Coordination for the Improvement of Higher Education Personnel [Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)] and Foundation to Support Research in Minas Gerais State [Fundação de Amparo a Pesquisa no Estado de Minas Gerais (FAPEMIG)] for financial support and for granting scholarships. References Bal, H.B., Das, S., Dangar, T.K., Adhya, T.K., 2013. ACC deaminase and IAA producing growth promoting bacteria from the rhizosphere soil of tropical rice plants. J. Basic Microbiol. 53, 972–984. Beneduzi, A., Peres, D., Vargas, L.K., Bodanese-Zanettini, M.H., Passaglia, L.M.P., 2008. Evaluation of genetic diversity and plant growth promoting activities of nitrogen-fixing bacilli isolated from rice fields in South Brazil. Appl. Soil Ecol. 39, 311–320. Cabala, R.P., Wild, A., 1982. Direct use of low grade phosphate rock Brasil as fertilizer: effect of reaction time in soil. Plant Soil 65, 351–362. CONAB (Companhia Nacional de Abastecimento). Acompanhamento da safra Brasileira.http://www.conab.gov.br/OlalaCMS/uploads/arquivos/ 13_07_09_09_04_53_boletim_graos_junho__2013. pdf. March 12, 2014. Costa, E.M., Nóbrega, R.S.A., Carvalho, F., Trochmann, A., Ferreira, L.V.M., Moreira, F. M.S., 2013. Plant growth promotion and genetic diversity of bacteria isolated from cowpea nodules. Pesq. Agropec. Bras. 48, 1275–1284. Crusciol, C.A.C., Mauad, M., Cassia, R., Alvarez, F., Lima, E.V., Tiritan, C.S., 2005. Phosphorus doses and root growth of upland rice. Bragantia 64, 643–649. Dias, P.R., Gatiboni, L.C., Ernani, P.R., Miquelluti, D.J., Chaves, D.M., Brunetto, G., 2014. Partial substitution of soluble phosphate by rock phosphate in the planting of Eucalyptus benthamii and Eucalyptus dunnii in southern Brazil. R. Bras. Ci. Solo 38, 516–523. Doll, E.C., Miller, H.F., Freeman, J.F., 1960. Initial and residual effects of rock phosphate and superphosphate. Agron. J. 52, 246–250.
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EMBRAPA, 2006. Sistema Brasileiro de Classificação de Solos, 2nd ed. Centro Nacional de Pesquisa de Solos, Rio de Janeiro, Brazil. Estrada, G.A., Baldani, V.L.D., De Oliveira, D.M., Urquiaga, S., Baldani, J.I., 2013. Selection of phosphate-solubilizing diazotrophic Herbaspirillum and Burkholderia strains and their effect on rice crop yield and nutrient uptake. Plant Soil 369, 115–129. Fageria, N.K., Baligar, V.C., Wright, R.J., 1991. Influence of phosphate rock sources and rates on rice and common bean production in an Oxisol. Plant Soil 45, 539–546. Ferreira, D.F., 2011. Sisvar: a computer statistical analysis system. Ciênc. Agrotecnol. 35, 1039–1042. Fred, E.B., Waksman, S.A., 1928. Laboratory Manual of General Microbiology: with Special Reference to the Microorganisms of the Soil. McGraw-Hill, New York. Habibi, S., Djedidi, S., Prongjunthuek, K., Mortuza, M.F., Ohkama-Ohtsu, N., Sekimoto, H., Yokoyoma, T., 2014. Physiological and genetic characterization of rice nitrogen fixer PGPR isolated from rhizosphere soils of different crops. Plant Soil 379, 51–66. Hamdali, H., Bouizgarne, B., Hafidi, M., Lebrihi, A., Virolle, M.J., Ouhdouch, Y., 2008. Screening for rock phosphate solubilizing Actinomycetes from Moroccan phosphate mines. Appl. Soil Ecol. 38, 12–19. Illmer, P., Schinner, F., 1992. Solubilization of inorganic phosphates by microorganisms isolated from forest soils. Soil Biol. Biochem. 24, 389–395. Korndorfer, G.H., Cabezas, W.A.L., Horowitz, N., 1999. Efficiency of reactive rock phosphates for corn production in Brazil. Sci. Agric. 56, 1–9. Krey, T., Baum, C., Ruppel, S., Seydel, M., Eichler-Löbermann, B., 2013. Organic and inorganic P sources interacting with applied rhizosphere bacteria and their effects on growth and p supply of maize. Commun. Soil Sci. Plant Anal. 44, 3205–3215. Lavakush, Yadav, J., Verma, J.P., Jaiswal, D.K., Kumar, A., 2014. Evaluation of PGPR and different concentration of phosphorus level on plant growth, yield and nutrient content of rice (Oryza sativa). Ecol. Eng. 62, 123–128. Léon, L.A., Fenster, W.E., Hammond, L.L., 1986. Agronomic potencial of eleven phosphate rocks from Brazil, Colombia, Perú and Venezuela. Soil Sci. Soc. Am. J. 50, 798–802. Lima, A.S., Nòbrega, R.S.A., Barberi, A., Silva, K., Ferreira, D.F., Moreira, F.M.S., 2009. Nitrogen-fixing bacteria communities occurring in soils under different uses in the Western Amazon region as indicated by nodulation of siratro (Macroptilium atropurpureum). Plant Soil 320, 1–19. Lin, T.F., Huang, H.I., Shen, F.T., Young, C.C., 2006. The protons of gluconic acid are the major factor responsible for the dissolution of tricalcium phosphate by Burkholderia cepacia CC-A174. Bioresour. Technol. 97, 957–960. Malavolta, E., Vitti, G.C., Oliveira, S.A., 1997. Avaliação do estado nutricional das plantas: princípios e aplicações. 2 ed. Piracicaba: Potafos. Marra, L.M., Oliveira, S.M., Soares, C.R.F.S., Moreira, F.M.S., 2011. Solubilisation of inorganic phosphates by inoculant strains from tropical legumes. Sci. Agric. 68, 603–609. Marra, L.M., Soares, C.R.F.S., Oliveira, S.M., Ferreira, P.A.A., Soares, B.L., Carvalho, R.F., Lima, J.M., Moreira, F.M.S., 2012. Biological nitrogen fixation and phosphate solubilization by bacteria isolated from tropical soils. Plant Soil 353, 289–307. Mehlich, A., 1953. Determination of P, Ca, Mg, K, Na and NH4 by North Carolina soil testing laboratories. University of North Carolina, Raleigh. Nautiyal, C.S., 1999. An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol. Lett. 170, 265–270. Novais, R.F., Smyth, T.J., 1999. Fósforo em solo e planta em condições tropicais. Viçosa, Universidade Federal de Viçosa, 399p. Oliveira-Longatti, S.M., Marra, L.M., Moreira, F.M.S., 2013. 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Phosphate-solubilising bacteria enhance Oryza sativa growth and nutrient accumulation in an oxisol fertilized with rock phosphate Elaine Martins da Costa, Wellington de Lima, Silvia M. Oliveira-Longatti, Fatima M. de Souza* Setor de Biologia, Microbiologia e Processos Biológicos do Solo, Departamento de Ciência do Solo, Universidade Federal de Lavras, Campus UFLA, 37200-000 Lavras, Minas Gerais, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 February 2015 Received in revised form 10 June 2015 Accepted 28 June 2015 Available online xxx
This study aimed to evaluate the ability of phosphate-solubilizing bacteria (PSB) to enhance growth and nutrient accumulation of rice plants grown in an Oxisol fertilized with Bayóvar rock phosphate (BRP). The experiment was entirely randomised and consisted of the following 12 treatments: individual inoculation with five PSB strains in pots containing BRP; a control with BRP without inoculation; five different soluble phosphorus (P) concentrations (50, 100, 150, 200 and 250 mg P2O5 dm 3) applied as NH4H2PO4 and a control without P or inoculation. The PSB strains UFLA 04-21 (Burkholderia sp.), UFLA 0310 (Paenibacillus kribbensis), UFPI B5-6 (Enterobacter sp.) and UFPI B5-8A (Pseudomonas sp.) increased the shoot dry matter, root dry matter and total dry matter, the number of tillers and the accumulation of phosphorus, nitrogen, calcium, magnesium, sulphur and boron compared with the treatment with BRP without inoculation. The increases in the dry matter of the shoots and roots were 52% and 120%, respectively, with the strain UFLA 03-10 (Paenibacillus kribbensis) inoculation treatment. The four strains promoted shoot dry matter that were equivalent to approximately 60% of the shoot dry matter produced in the treatment with 150 mg dm 3 of soluble P and increased the P accumulation in the shoots compared with the treatments with 50 and 100 mg P2O5 dm 3 of soluble P. These results indicate that inoculation with PSB combined with rock phosphate is an economical and sustainable strategy for improving the growth and nutrient accumulation of the rice plants. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Plant growth promoting rhizobacteria Biofertilizers Bayóvar rock phosphate
1. Introduction Phosphorus (P) is one of the most limiting nutrients for crop development in tropical soils because of its complex dynamics. In addition to the low natural content available for uptake by plants, most of the P applied to the soil is retained in its particles because of the adsorption and precipitation reactions that are favoured by the acidity and oxidic mineralogy of most of these soils producing inorganic phosphates (Novais and Smyth, 1999). Thus, high doses of phosphate fertilisers are required to obtain satisfactory yields. Because of the potential risk of depleting natural reserves of soluble phosphates (SPs), the high cost and the residual effect of SPs when applied in large amounts, the use of alternative P sources, such as less concentrated rock phosphates (RPs) that are lower in cost, has been suggested (Fageria et al., 1991; Korndorfer et al., 1999; Léon et al., 1986). However, RPs dissolve more slowly than SPs, and the dissolution varies between the different types of
* Corresponding author: Fax: +55 35 3829 12 51. http://dx.doi.org/10.1016/j.ecoleng.2015.06.045 0925-8574/ ã 2015 Elsevier B.V. All rights reserved.
commercially available RPs. Typically, igneous and metamorphic RPs, such as Brazilian RPs, demonstrate low reactivity, while sedimentary phosphates are more reactive and, therefore, have been used more frequently in agriculture (Dias et al., 2014; Léon et al., 1986; Resende et al., 2006). The more reactive RPs include Bayóvar rock phosphate (BRP), which is of sedimentary origin and is formed by the deposition and subsequent decomposition of marine animal remains from the Piura region (province of Sechura) in Peru (Léon et al., 1986). This phosphate containing approximately 28% P2O5 and 31% calcium, is insoluble in water and is approximately 13% soluble in citric acid. In Brazil, few studies on BRP have been conducted, but it has been shown that this phosphate can partially replace the use of SPs on agricultural crops, indicating its potential use in agriculture (Dias et al., 2014). An important method for increasing the utilisation efficiency of RPs and/or reducing the use of SPs fertilisers is the use of bacteria that solubilise inorganic phosphates and could potentially increase the amount of P available to plants (Estrada et al., 2013; Lavakush et al., 2014; Oliveira-Longatti et al., 2013, 2014; Pereira and Castro, 2014; Shankar et al,. 2011). These bacteria participate in the
E. Martins da Costa et al. / Ecological Engineering 83 (2015) 380–385
solubilisation process through various mechanisms. The mechanism most often cited in the literature is the production and release of low molecular weight organic acids. However organic acid production does not always correlate with the solubilisation of inorganic phosphates (Marra et al., 2012). Other reported mechanisms include extrusion of protons via cellular respiration and the uptake of ammonium as a nitrogen source (Illmer and Schinner, 1992), exopolysaccharide production (Yi et al., 2008) and siderophore production (Hamdali et al., 2008). In Brazil, rice (Oryza sativa L.) is one of the main cereals grown, with an estimated planted area of approximately 2427,000 hectares and an estimated production of 12 million tons for the years 2011–2012 (CONAB, 2013). Rice grown under rainfed systems, despite occupying approximately two-thirds of the total cultivated area, accounts for approximately one-third of the national yield (Crusciol et al., 2005). This low production is partially due to the fact that most of this rice is grown in Cerrado soils, which tend to have low P availability in most cases. Therefore, to achieve high productivity, the crop requires high doses of this nutrient, which results in higher production costs. The combined use of low cost RPs and inoculation with phosphate-solubilising bacteria (PSB) is an alternative technique to improve the nutrition, growth and yield of rice and to partially offset the cost of SP fertilisers. Furthermore, some PSB also contribute to the growth and nutrition of crops by participating in other biological processes (Costa et al., 2013; Oliveira-Longagatti et al., 2013, 2014). In general, few studies investigating the combined application of PSB and RPs have been reported, and there are no reports of this type for rice crop grown in Brazilian soils. Thus, the aim of this study was to evaluate the growth and nutrient accumulation of rice plants inoculated with inorganic phosphatesolubilising bacteria and grown in pots containing Oxisol fertilized with Bayóvar rock phosphate. 2. Materials and methods 2.1. Strains evaluated Five strains of bacteria belonging to the collection of the Laboratory of Soil Microbiology, UFLA (Universidade Federal de Lavras—Federal University of Lavras) were used in this study. The origin, accession numbers of 16S rRNA gene sequence and plant growth promoting characteristics of these strains are shown in Table 1.
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2.2. Count of calcium phosphate-solubilising bacteria in the soil Prior to the experiment, a single soil sample was collected from each pot to form a composite sample and to estimate the number of calcium phosphate-solubilising bacteria in the soil. Serial dilutions of the soil sample up to 10 7 were prepared in saline (8,5 g L 1 NaCl). Streaking with Drigalski spatula was performed using 100 mL of the dilution suspensions on plates containing NBRIP culture medium (Nautiyal, 1999) which were incubated at 28 C. Counts were periodically conducted until the eighth day after inoculation. 2.3. Experiment in greenhouse The experiment was conducted in a greenhouse from December 2013 to March 2014. Polypropylene pots with a 4 dm3 capacity that contained yellow–red Latosol (Oxisol) (EMBRAPA, 2006) with a clayey texture and a low P content, collected from the 0–20 cm layer in Lavras, Minas Gerais. Table 2 lists the chemical and physical characteristics of the soil before liming and fertilisation. Based on the chemical analysis of the soil, liming was performed to increase the base saturation to 50% using calcium carbonate (CaCO3) and magnesium carbonate (MgCO3) at a 4:1 ratio. The soil was incubated for 40 days with a moisture of approximately 60% of the total pore volume. The experimental design was completely randomised with four replicates. The following 12 treatments were used in this study: individual inoculation with five PSB strains (UFLA 04-21, UFLA 04155, UFLA 03-10, UFPI B5-6 and UFPI B5-8A) in pots with soil containing Bayóvar RP (150 mg P2O5 dm 3); a control with Bayóvar RP (150 mg P2O5 dm 3) without inoculation; five different concentrations of soluble phosphate (50, 100, 150, 200 and 250 mg P2O5 dm 3) applied as monoammonium phosphate (MAP) (NH4H2PO4) and a control without P or inoculation. Fertilisation was conducted as follows for all treatments (mg dm 3): 450 of N, 350 of K and 40 of S, provided as Urea [(NH2)2CO], Potassium chloride (KCl) and potassium sulphate (K2SO4), respectively, and 1.5 of Cu, 3.6 of Mn, 5.0 of Zn, 0.8 of B and 0.15 of Mo provided as copper sulphate (CuSO45H2O), manganese chloride (MnCl24H2O), zinc sulphate (ZnSO47H2O), boric acid (H3BO3) and ammonium molybdate [(NH4)6Mo7O244H2O], respectively. The calculations to determine the N dose took into consideration the amounts already provided by NH4H2PO4 at the various P concentrations. The nitrogen and potassium fertilisation treatments were divided into five applications, the first occurred at
Table 1 Origin and plant growth promoting characteristics of the strains evaluated. Strains
Location/ LUS
Phylogenetic affiliation
Accession No. in GenBank of 16S rRNA sequences (NCBI)
Plant growth promoting characteristics(c,d,e) IAA in DYGS Free-living nitrogen medium fixation (mg ml 1)* Tryptophan
In vitro solubilization
CaHPO4 AlH6P3O12 FePO4.2H2O – UFLA 0421 UFLA 04155 UFLA 0310 UFPI B5-6 UFPI B58A
+
AM/AG
Burkholderia sp..(a)
FJ534643
+
+
ND
2.53 2.14
+
AM/SFI
Burkholderia fungorum (b) Paenibacillus kribbensis (c) Enterobacter sp.(d) Pseudomonas sp.(d)
GU144370
+
+
ND
6.29 4.53
+
JQ041885
+
–
+
ND
ND
KJ879611 KJ879613
+ +
– –
– –
2.87 26.54 – 5.40 9.71 –
MG/STF PI/AG PI/AG
ND
LUS—Land-use systems; AM—Amazônia state; MG—Minas Gerais estate; PI—Piauí estate; AG—Agriculture; SFI—Secondary forest in an initial stage of regeneration; STF— Semideciduous tropical forest; ND—Not determined; *Production of indole-3-acetic acid (IAA) in DYGS medium without ( ) and with (+) tryptophan. (a)Lima et al., 2009; (b) Silva et al., 2012; (c)Marra et al., 2012; (d)Costa et al. unpublished data; (e)Oliveira-Longatti et al., 2013.
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Table 2 Chemical and physical characteristics of soil sample before implantation of the experiment. Depth cm
pH H2O
Pa mg dm
K+
Ca2+ cmolc dm
0–20
5.4
0.84
Depth cm
O.M %
P-rem ml L 1
Zn mg dm
0–20
2.11
3.54
1.59
3
24.00
Mg2+
Al3+
H+Al
TB
ECE
T
V %
m
0.10
0.10
2.90
1.86
1.96
4.76
39.11
5.10
3
1.70 Fe
Mn
Cu
B
S
Sand %
Silt
Clay
29.78
4.98
3.15
0.13
28.07
17
10
73
3
a
Mehlich-1 method; TB—Total base cations; ECE—Effective cation exchange capacity; T—Potential cation exchange capacity; V—index of base saturation; m—index of aluminium saturation; O.M—Organic matter.
moistened filter paper and cotton, where they remained for 72 h in an incubator at 28 C until the emergence of rootlets. Six seeds were sown per pot. Eight days after emergence, thinning to two seedlings was performed. The bacterial strains were cultured in liquid medium 79 (Fred and Waksman, 1928) under stirring of 110 rpm at 28 C for 48 h. For each inoculated treatment, 1 mL of
planting followed by applications 30, 45, 60 and 75 days after planting. Before planting, rice seeds (cultivar BRS-MG Relâmpago, recommended for rainfed farming systems) were surface sterilised using 98% ethanol (30 s), 2% sodium hypochlorite (two minutes) and subsequent successive washes in sterile distilled water. Next, the seeds were placed in sterilised Petri dishes containing
70
a
16
a b
TDM SDM TN
c
g pot
-1
c d
10 d c
c d
d
20
e e
10
d
a
d
8 a
b
d
d d
e
e
b
c
d
30
12
b
c
-1
50
40
14
a a
d
c
6
c
Tillers number pot
60
4 e 2
f f
200
150
250 Solu ble P
c
ble P
c
Solu
d
P 10 0
d
Solu ble P
d
Solu ble
d
Solu ble P
hout nd w it out I a
e
b b a
UFLA
30
e
150 mg
P
g pot
-1
20
W ith
10
50
0 f
BRP 04-1 55 + 150 BRP UFLA 03-1 0+1 50 B RP UFLA 04-2 1+1 50 B RP UF P I B58A + 150 BRP UFP I B56+1 50 B RP
0
40
50
RDM
60
250 ble P Solu
200
P 15 0
P 10 0
Solu ble P
Solu ble
Solu ble
P 50 Solu ble
BRP 04-1 55 + 150 BRP UFLA 03-1 0+1 50 B RP UFLA 04-2 1+1 50 B RP UF P I B58A + 150 BRP UF P I B56+1 50 B RP
150 mg
UFLA
W ith
out I a
nd w it
hout
P
70
Fig. 1. Total dry matter (TDM), shoot dry matter (SDM), tillers number (TN) and root dry matter (RDM) of rice plants (Oryza sativa L.) grown under different conditions of phosphorus supply for 90 days after planting in pots with a Oxisol. Means followed by the same letter do not differ by the Scott–Knott test at a 5% probability. BRP: Bayóvar rock phosphate (mg P2O5 dm 3) without inoculation; soluble P = Monoammonium phosphate (mg P2O5 dm 3); UFLA 04-155 (Burkholderia fungorum); UFLA 04-21 (Burkholderia sp.); UFLA 03-10 (Paenibacillus kribbensis); UFPI B5-6 (Enterobacter sp.); UFPI B5-8A (Pseudomonas sp.).
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inoculum at a concentration of 1 108 bacterial cells mL added for each seed sown.
1
was
2.4. Plant sampling and analysis Plants were harvested 90 days after planting, and the number of tillers per plant (NT) was assessed. Then, the shoots were washed with distilled water. The plant material was dried in a convection oven at 60 C to determine the shoot dry matter (SDM), root dry matter (RDM), total dry matter (TDM) and the accumulation of macronutrients [nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and sulphur (S)] and micronutrients [copper (Cu), iron (Fe), manganese (Mn), zinc (Zn) and Boron (B)] in the shoots (Malavolta et al., 1997). 2.5. pH and phosphorus available in the rhizospheric soil after the experiment After collecting the plants, rhizospheric soil samples were taken from the pots of each treatment group to determine the pH (H2O) and the available P extracted using the Mehlich-1 method (Mehlich, 1953). 2.6. Statistical analysis According to the Shapiro–Wilks test, the test data were normally distributed and were subjected to analysis of variance (ANOVA), using the statistical analysis program Sisvar 5.3 (Ferreira, 2011). The effects of the treatments were compared by the Scott– Knott test at P < 0.05. 3. Results 3.1. Count of calcium phosphate-solubilising bacteria in the soil Calcium phosphate-solubilising bacteria were detected in the studied soil up to a dilution of 10 3. The estimated number of these organisms was 2.1 104 CFU g 1 soil. 3.2. Influence of PSB inoculation on growth and nutrient accumulation in rice plants The treatments had a significant effect on the production of SDM, RDM and TDM, on the NT (Fig. 1) and on the accumulation of macro- and micro-nutrients (Table 3). Four (UFLA 04-21, UFLA 03-
383
10, UFPI B5-6 and UFPI B5-8A) of the five strains promoted the production of SDM, RDM and TDM and NT to similar levels that were greater than those observed for the treatment with Bayóvar RP without inoculation (Fig. 1). The SDM increased to 42% and 52% in the treatments inoculated with UFLA 04-21 (Burkholderia sp.) and UFLA 03-10 (Paenibacillus kribbensis), respectively, compared with the treatment with Bayóvar RP without inoculation. Greater increases of 65%, 105%, 111% and 120% in the RDM were observed in the treatments inoculated with UFPI B5-8A (Pseudomonas sp.), UFLA 04-21 (Burkholderia sp.), UFPI B5-6 (Enterobacter sp.) and UFLA 03-10 (Paenibacillus kribbensis), respectively. However, compared with the treatments with SP, these strains produced lower yields of SDM, RDM and TDM; they only produced more tillers than the treatment with 50 mg P2O5 dm 3 (Fig. 1). The strain UFLA 04-155 yielded results that were similar to those of the treatment with BRP without inoculation for all the growth parameters assessed. Comparison of the treatments with different concentrations of SP revealed that applying 200 mg P2O5 dm 3 is sufficient for promoting maximal rice plant growth (Fig. 1). In this treatment, the SDM production and NT results were similar to the results from the treatment with the highest SP concentration (250 mg P2O5 dm 3), and the production of RDM and TDM were greater than that of the treatment with the highest SP concentration. Regarding the accumulation of P in shoots, the treatments inoculated with the strains UFLA 04-21, UFLA 03-10, UFPI B5-6 and UFPI B5-8A yielded significantly higher accumulations of P than the treatment with Bayóvar RP without inoculation and the treatments with 50 and 100 mg P2O5 dm 3 SP (Table 3). The increase in P accumulation in the shoot was 13% and 39% for the treatments inoculated with the strains UFPI B5-8A and UFLA 03-10, respectively, compared with the treatment with Bayóvar RP without inoculation. Most notably, the strain UFLA 03-10 yielded a P accumulation in the shoots that was similar to P accumulation resulting from the treatments with 200 and 250 mg P2O5 dm 3 SP. Significant increases in the accumulation of the other macronutrients in the shoots (N, K, Ca, Mg and S) were also observed for the treatments inoculated with the strains UFLA 04-21, UFLA 0310, UFPI B5-6 and UFPI B5-8A, with the exception of K accumulation for the treatment inoculated with the strains UFPI B5-6 and UFPI B5-8A, which were similar to the treatment with BRP without inoculation (Table 3). All of the treatments with BRP yielded significant increases in Ca accumulation compared with the treatments with SP. Most notably, the inoculation treatments with the strains UFLA 03-10, UFPI B5-8A and UFLA 04-21 yielded
Table 3 Accumulation of macronutrients and micronutrients in shoots of rice plants (Oryza sativa L.) grown under different conditions of phosphorus supply for 90 days after planting in pots with a Oxisol. Treatments Without I Without P 150 mg P2O5 dm 3 (BRP) UFLA 04-155 + 150 mg P2O5 dm 3(BRP) UFLA 04-21 + 150 mg P2O5 dm 3(BRP) UFLA 03-10 + 150 mg P2O5 dm 3(BRP) UFPI B5-6 + 150 mg P2O5 dm 3 BRP) UFPI B5-8A + 150 mg P2O5dm 3 (BRP) 50 mg P2O5dm 3 (MAP) 100 mg P2O5 dm 3 (MAP) 150 mg P2O5 dm 3 (MAP) 200 mg P2O5 dm 3 (MAP) 250 mg P2O5 dm 3 (MAP) CV (%)
N (mg pot 8.88 f 326.05 e 302.10 e 431.97 d 490.38 c 440.55 d 405.30 d 404.28 d 520.62 c 631.48 b 766.58 a 758.85 a 7.22
P 1
K
Ca
Mg
S
) 0.09 e 27.16 c 25.09 c 34.21 b 37.73 a 34.68 b 30.59 b 19.02 d 26.35 c 31.39 b 40.88 a 36.57 a 11.08
3.15 c 244.32 b 221.27 b 350.90 a 405.19 a 286.51 b 249.15 b 389.45 a 449.07 a 421.66 a 469.15 a 391.93 a 16.56
0.74 f 60.93 c 55.08 c 86.05 a 92.74 a 73.16 b 87.60 a 13.60 e 31.95 d 39.28 d 34.60 d 40.98 d 10.30
1.58 f 31.78 e 28.56 e 50.74 d 47.57 d 45.21 d 43.50 d 46.39 d 59.27 c 66.82 c 83.17 a 73.63 b 12.08
0.82 e 46.45 d 40.24 d 60.64 c 61.65 c 63.28 c 59.04 c 53.67 c 70.93 b 75.51 b 82.84 a 73.78 b 10.58
Cu (mg pot 2.50 e 173.90 d 152.33 d 211.99 c 209.03 c 225.20 b 183.45 d 231.56 b 234.90 b 272.14 a 296.13 a 270.34 a 8.94
1
Fe
Mn
Zn
B
742.5 f 1035.68 e 1422.38 d 1506.36 d 1405.33 d 1193.18 e 1336.92 d 1682.71 d 2333.88 c 2371.87 c 3439.03 b 3873.08 a 12.06
113.50 c 8616.05 b 7959.65 b 9614.68 a 8756.58 b 8391.23 b 9194.36 a 9583.97a 9559.88 a 9803.06 a 9175.58 a 8555.49 b 6.97
17.00 f 2554.89 d 1701.77 e 3880.50 c 3244.02 d 3727.41 c 3097.00 d 4115.73 c 5433.35 b 5734.74 b 6722.34 a 5956.12 b 9.84
5.75 e 194.72 d 168.91 d 318.92 b 248.79 c 283.86 b 370.41 a 379.93 a 366.68 a 397.93 a 378.44 a 386.20 a 10.28
)
BRP—Bayóvar rock phosphate; MAP—Monoammonium phosphate; UFLA 04-155 (Burkholderia fungorum); UFLA 04-21 (Burkholderia sp.); UFLA 03-10 (Paenibacillus kribbensis); UFPI B5-6 (Enterobacter sp.); UFPI B5-8A (Pseudomonas sp.). Values in the same column with different letters are significantly different by the Scott–Knott test (P < 0.05).
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participate in other processes that promote plant growth (Table 1). However, we emphasise that it is not always possible to correlate in vitro activity with in vivo effects on plant growth and nutrition. For example, despite its ability to solubilise insoluble inorganic phosphates in vitro, the strain UFLA 04-155 (Burkholderia fungorum) did not appear to promote the growth of rice plants in the present study. Of the macro- and micro-nutrients assessed, this strain only increased the accumulation of Fe. Strains belonging to the genera Burkholderia, Enterobacter, Paenibacillus and Pseudomonas are often described as potential promoters of plant growth because of their ability to participate in various biological processes. In rice crops, significant increases in the growth of shoots and roots were observed in plants inoculated with strains of Burkholderia (Estrada et al., 2013; Souza et al., 2013), Enterobacter (Habibi et al., 2014; Shankar et al., 2011), Paenibacillus (Bal et al., 2013; Beneduzi et al., 2008) and Pseudomonas (Habibi et al., 2014; Lavakush et al., 2014; Yasmin et al., 2004). However, only two of these previous studies (Estrada et al., 2013; Lavakush et al., 2014) evaluated the contribution of phosphate solubilisation to the growth and nutrition of rice in pots with non-sterile soil. Estrada et al., (2013) demonstrated that inoculation with a Burkholderia vietnamiensis strain increased SDM production, P and N accumulation and the yield of rice grown in pots with soil fertilised with tricalcium phosphate [Ca3(PO4)2]. The combination of the Pseudomonas, Azotobacter chroococcum and Azospirillum brasilense strains and 30 kg ha 1 P2O5 (unnamed source) in an experiment conducted in pots that contained soil resulted in greater plant growth, greater accumulation of N, P and K and a greater yield of rice compared with the treatment without inoculation with 30 kg ha 1 P2O5 (Lavakush et al., 2014). The ability of strains belonging to the genera Enterobacter and Paenibacillus to solubilise calcium phosphate has been reported in several previous studies (Beneduzi et al., 2008; Costa et al., 2013; Krey et al., 2013; Marra et al., 2012; Shankar et al., 2011). The contribution of calcium phosphate solubilisation to growth and plant nutrition has also been reported in grasses, such as maize inoculated with strains belonging to the Enterobacter genus (Krey et al., 2013). However, our study is the first to report the contribution of strains belonging to the genera Enterobacter and Paenibacillus to the growth and nutrition of rice crops through the solubilisation of inorganic rock phosphate. According to the literature, lowering the pH of the solution by releasing the organic acids in the rhizosphere is one of the main mechanisms responsible for the solubilisation of insoluble inorganic phosphates in soils (Lin et al., 2006). However, the soil solution pH did not change after the experiment for any of the treatments in the present study. This result is consistent with those of Marra et al., (2011), who demonstrated that the solubilisation of
52%, 44% and 41% greater Ca accumulation, respectively, compared with the treatment with BRP without inoculation. The accumulation of micro-nutrients in the shoots varied depending on the treatment. Inoculation with the strain UFLA 04155 only yielded a greater accumulation of Fe, while inoculation with the strain UFLA 04-21 resulted in a greater accumulation of all the micro-nutrients assessed (Cu, Fe, Mn, Zn and B) compared with the treatment with Bayóvar RP without inoculation (Table 3). The strain UFLA 03-10 yielded a greater accumulation of Cu, Fe and B. The strain UFPI B5-6 increased the accumulation of Cu, Zn and B. The strain UFPI B5-8A increased the accumulation of Fe, Mn and B. The treatment with UFLA 04-155 inoculation exhibited results that were similar to the results for the treatment with only Bayóvar RP in terms of all of the macro-nutrients and most of the micronutrients (Cu, Mn and B) accumulated (Table 3). 3.3. pH (H2O) and available phosphorus in the rhizospheric soil after the experiment The pH (H2O) values of the soil did not vary significantly among the various treatments after the experiment. However, the treatments significantly affected the level of P available in the rhizospheric soil (Table 4). The treatment with Bayóvar RP without inoculation resulted in a significantly higher level of available P (12.93 mg dm 3) than the other treatments. The treatments including inoculation with the UFLA 04-21 and UFPI B5-8A strains yielded similar values (11.54 and 11.08 mg of P dm 3) that were greater than those for the remaining strains (Table 4). 4. Discussion The combined use of low-cost RPs and inoculation with PSB is an important strategy for reducing the use of SP fertilisers and production costs while increasing the sustainability of agroecosystems. Of the five bacterial strains evaluated in this study, four contributed to the growth and nutrition of rice plants by solubilising the inorganic calcium phosphate with soil containing Bayóvar RP. Strains belonging to the genera Burkholderia (UFLA 0421), Enterobacter (UFPI B5-6), Paenibacillus (UFLA 03-10) and Pseudomonas (UFPI B5-8A) were able to significantly increase the production of SDM, RDM and TDM, the NT and the accumulation of P, N, Ca, Mg, S and B compared to the treatment with Bayóvar RP without inoculation. Although the studied soil contained a native population of calcium phosphate-solubilising bacteria of approximately 2.1 104 CFU g 1 of soil, the four studied strains mentioned were more efficient. In previous studies, these strains have demonstrated the ability to solubilise insoluble inorganic phosphates in vitro and to
Table 4 pH (H2O) of the rhizosphere soil samples and available phosphorus (P) from each treatment after the conducting experiment. Treatments
pH (H2O)
Available P (Mehlich-1) (mg dm
Without I Without P 150 mg P2O5 dm 3 (BRP) UFLA 04-155 + 150 mg P2O5 dm 3 (BRP) UFLA 04-21 + 150 mg P2O5 dm 3 (BRP) UFLA 03-10 + 150 mg P2O5 dm 3 (BRP) UFPI B5-6 + 150 mg P2O5 dm 3 (BRP) UFPI B5-8A + 150 mg P2O5 dm 3 (BRP) 50 mg P2O5 dm 3 (MAP) 100 mg P2O5 dm 3 (MAP) 150 mg P2O5 dm 3 (MAP) 200 mg P2O5 dm 3 (MAP) 250 mg P2O5 dm 3 (MAP) CV (%)
4.7 a 4.9 a 4.8 a 4.8 a 4.9 a 4.9 a 5.1 a 4.8 a 4.9 a 4.9 a 4.8 a 4.9 a 1.91
0.63 f 12.93 a 7.39 c 11.54 b 7.76 c 5.14 d 11.02 b 0.56 f 1.27 f 1.71 f 1.56 f 3.20 e 15.78
3
)
RP—Bayóvar rock phosphate; MAP—Monoammonium phosphate; UFLA 04-155 (Burkholderia fungorum); UFLA 04-21 (Burkholderia sp.); UFLA 03-10 (Paenibacillus kribbensis); UFPI B5-6 (Enterobacter sp.); UFPI B5-8A (Pseudomonas sp.). Values in the same column with different letters are significantly different by the Scott–Knott test (P < 0.05).
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insoluble inorganic phosphates is not always associated with the lowering of pH. We found the highest levels of available P in the soil solutions treated with BRP and the lowest levels in the soil solutions treated with SP. This finding may be due to the extraction method used (Mehlich, 1953), which despite its standard use for assessing the availability of P in soils fertilised with soluble sources, may be inappropriate for soils fertilised with RPs because this method begins with the extraction process of solubilisation by H+ ions, possibly promoting the excessive dissolution of particles of RPs and thus overestimating the levels of available P (Doll et al., 1960; Cabala and Wild, 1982). In our study, the soluble P levels resulting from the treatments with BRP were most likely overestimated because there was no observed relationship between the levels in the soil and the amount of P that accumulated in the shoots. Considering the potential risk of depleting more naturally concentrated phosphate reserves and the high cost of SP fertilisers, it is important to use alternate methods, such as those used in our study, that at least partially replace SPs. In this case, the combined use of BRP and inoculation with bacterial strains that efficiently solubilise insoluble phosphates was shown to be a good strategy. The strains that contributed to the growth and nutrition of rice plants in the present study will be evaluated under field conditions in future studies to verify their potential as crop inoculants. 5. Conclusions Inoculation of rice with strains UFLA 04-21 (Burkholderia sp.), UFPI B5-6 (Enterobacter sp.), UFLA 03-10 (Paenibacillus kribbensis) and UFPI B5-8A (Pseudomonas sp.) enhanced plant growth and nutrient accumulation by solubilization of insoluble calcium phosphate. These four strains may be used as biofertilizers, constituting an economical and sustainable strategy to increase rice yield. Acknowledgments The authors thank the National Council for Scientific and Technological Development [Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)], the Coordination for the Improvement of Higher Education Personnel [Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)] and Foundation to Support Research in Minas Gerais State [Fundação de Amparo a Pesquisa no Estado de Minas Gerais (FAPEMIG)] for financial support and for granting scholarships. References Bal, H.B., Das, S., Dangar, T.K., Adhya, T.K., 2013. ACC deaminase and IAA producing growth promoting bacteria from the rhizosphere soil of tropical rice plants. J. Basic Microbiol. 53, 972–984. Beneduzi, A., Peres, D., Vargas, L.K., Bodanese-Zanettini, M.H., Passaglia, L.M.P., 2008. Evaluation of genetic diversity and plant growth promoting activities of nitrogen-fixing bacilli isolated from rice fields in South Brazil. Appl. Soil Ecol. 39, 311–320. Cabala, R.P., Wild, A., 1982. Direct use of low grade phosphate rock Brasil as fertilizer: effect of reaction time in soil. Plant Soil 65, 351–362. CONAB (Companhia Nacional de Abastecimento). Acompanhamento da safra Brasileira.http://www.conab.gov.br/OlalaCMS/uploads/arquivos/ 13_07_09_09_04_53_boletim_graos_junho__2013. pdf. March 12, 2014. Costa, E.M., Nóbrega, R.S.A., Carvalho, F., Trochmann, A., Ferreira, L.V.M., Moreira, F. M.S., 2013. Plant growth promotion and genetic diversity of bacteria isolated from cowpea nodules. Pesq. Agropec. Bras. 48, 1275–1284. Crusciol, C.A.C., Mauad, M., Cassia, R., Alvarez, F., Lima, E.V., Tiritan, C.S., 2005. Phosphorus doses and root growth of upland rice. Bragantia 64, 643–649. Dias, P.R., Gatiboni, L.C., Ernani, P.R., Miquelluti, D.J., Chaves, D.M., Brunetto, G., 2014. Partial substitution of soluble phosphate by rock phosphate in the planting of Eucalyptus benthamii and Eucalyptus dunnii in southern Brazil. R. Bras. Ci. Solo 38, 516–523. Doll, E.C., Miller, H.F., Freeman, J.F., 1960. Initial and residual effects of rock phosphate and superphosphate. Agron. J. 52, 246–250.
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