Protoplasma DOI 10.1007/s00709-017-1112-1

ORIGINAL ARTICLE

Contribution of native phosphorous-solubilizing bacteria of acid soils on phosphorous acquisition in peanut (Arachis hypogaea L.) Madhusmita Pradhan 1 & Ranjan Kumar Sahoo 2 & Chinmay Pradhan 3 & Narendra Tuteja 4 & Santanu Mohanty 5

Received: 30 September 2016 / Accepted: 10 April 2017 # Springer-Verlag Wien 2017

Abstract The present investigation analyzes the in vitro P solubilization [Ca-P, Al-P, Fe(II)-P, and Fe(III)-P] efficiency of native PSB strains from acid soils of Odisha and exploitation of the same through biofertilization in peanut (Arachis hypogaea L.) growth and P acquisition. One hundred six numbers of soil samples with pH ≤ 5.50 were collected from five districts of Odisha viz., Balasore, Cuttack, Khordha, Keonjhar, and Mayurbhanj. One bacterial isolate from each district were selected and analyzed for their P solubilization efficiency in National Botanical Research Institute Phosphate broths with Ca, Al, and Fe-complexed phosphates. CTC12 and KHD08 transformed more amount of soluble P from Ca-P (CTC12 393.30 mg/L; KHD08 465.25 mg/L), Al-P (CTC12 40.00 mg/L; KHD08 34.50 mg/L), Fe(III)-P (CTC12 175.50 mg/L; KHD08 168.75 mg/L), and Fe(II)-P (CTC12 47.40 mg/L; KHD08 42.00 mg/L) after 8 days of incubation. The bioconversion of P by all the five strains in the broth medium followed the order Ca-P > Fe(III)-P >

Fe(II)-P > Al-P. The identified five strains were Bacillus cereus BLS18 (KT582541), Bacillus amyloliquefaciens CTC12 (KT633845), Burkholderia cepacia KHD08 (KT717633), B. cepacia KJR03 (KT717634), and B. cepacia K1 (KM030037) and further studied for biofertilization effects on peanut. CTC12 and KHD08 enhanced the soil available P around 65 and 58% and reduced the amount of each Al3+ about 79 and 81%, respectively, over the uninoculated control pots in the peanut rhizosphere. Moreover, all tested PSB strains could be able to successfully mobilize P from inorganic P fractions (non-occluded Al-P and Fe-P). The strains CTC12 and KHD08 increased the pod yield (114 and 113%), shoot P (92 and 94%), and kernel P (100 and 101%), respectively, over the control. However, B. amyloliquefaciens CTC12 and B. cepacia KHD08 proved to be the potent P solubilizers in promoting peanut growth and yield. Keywords PSB . Inorganic phosphates . P fraction . Peanut

Handling Editor: Néstor Carrillo * Santanu Mohanty [email protected] 1

Department of Soil Science and Agricultural Chemistry, College of Agriculture, OUAT, Bhubaneswar, Odisha, India

2

Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna AsafAli Marg, New Delhi, India

3

PG Department of Botany, Utkal University, Bhubaneswar, Odisha, India

4

Amity Institute of Microbial Technology, Amity University, Noida, India

5

Orissa University of Agriculture and Technology, Bhubaneswar, India

Introduction Phosphorous is the most vital macronutrient (Saber et al. 2005) after nitrogen which takes part in energy transfer, cell division, photosynthesis, biological oxidation, and other physiological metabolisms like respiration, energy storage, cell enlargement, vegetative growth, early root formation and growth, seed formation, etc. (Sashidhar and Podile 2010). P is also the primary prerequisite for seed formation and for nodule formation (Shenoy and Kalagudi 2005; Sagervanshi et al. 2012). In most agricultural soils, low solubility and poor mobility of soil P are of concern which very often limit plant growth (Gulati et al. 2008). Usually less than 5% soil P in the form of

M. Pradhan et al.

HPO42− and H2PO4− is absorbed by the plants (Rendig and Taylor 1989). Most of the inorganic phosphates are complexed with aluminum and iron [Al(OH)2H2PO4 or AlPO4· 2H2O; Fe(OH)2H2PO4 or FePO4·2H2O; Fe3(PO4)2·8H2O] in acidic soils which are highly stable compounds and thus become unavailable for plant uptake (Mohsin et al. 1995; Fearnside 1998; Richardson 2001; Bashan et al. 2013; Merbach et al. 2010). Consequently, to enhance P availability in acidic soils, heavy doses of inorganic P fertilizers are applied in agricultural lands which often get complexed with Al and Fe making it unavailable for plants. These complexes although hard to dissolve can be efficiently solubilized by certain microflora present in the rhizosphere soil. The fact is previously known and has been reported by several authors (Goldstein 2007; Jorquera et al. 2008). These beneficial microorganisms perform the release of bound phosphates as orthophosphates and facilitate the uptake of P to plants (Tripura et al. 2007; Anzuay et al. 2015). Different genera such as Pseudomonas, Bacillus, Rhizobium, Agrobacterium, Burkholderia, Acromobacter, Micrococcus, Aerobacter, Flavobacterium, Erwinia, Pantoea, Acinetobacter, Acetobacter, Enterococcus, Enterobacter, Mycobacterium, Serratia, Bradyrhizobium, and Azotobacter have been found to solubilize phosphate (Antoun et al. 1998; Kumar and Narula 1999; Maheshkumar et al. 1999; Rodríguez and Fraga 1999; Goldstein 2001; Pérez et al. 2007; Tripura et al. 2007; Ogut et al. 2010; Walpola and Yoon 2013; Kaur and Sudhakara Reddy 2014). An interdependent relationship always exists between bacteria that provide soluble phosphates and plant roots that supply carbon compounds (Pérez et al. 2007). Thus, to improve P nutrition in agricultural lands, an ecofriendly approach can be made by using the native phosphate-solubilizing bacteria. According to Richardson et al. (2001) and Mohammadi et al. (2011), application of PSBs enhanced several yield attributing parameters leading to significant increase in productivity of various crops. In peanuts, significant beneficial effects with application of Psolubilizing bacteria were also reported by Taurian et al. (2010) and Anzuay et al. (2015). Peanut (Arachis hypogaea L.) is an important oilseed crop as well as cash crop of tropical and subtropical regions of the world. Although India leads in groundnut production, the productivity is quite poor owing to traditional cultivation practices, dry belts, poor soil fertility, erratic rainfall, and poor input levels (Basu et al. 2008). Peanut occupies 30.5% of the total oil seed area in Odisha and accounts for 64.4% of the total oil seed production. The crop is grown throughout the year in three crop seasons, i.e., kharif, rabi, and rabi-summer in 2.43 lakh hectare with a production of 3.98 lakh tons and productivity of 1639 kg/ha (Parida et al. 2011). Although physical and textural conditions of acid lateritic soils (Alfisol) of the peanut-growing areas of Odisha are advantageous, the chemical characteristics (more

than 70% of the cropped area below pH 5.50) such as toxicity of aluminum (Al), low organic matter, and poor supply of calcium (Ca), magnesium (Mg), and phosphorous (P) are the main causes of low productivity (Karmakar et al. 1997; Raychaudhury et al. 2003). Thus, the present investigation is focused on isolation of native strains of phosphatesolubilizing bacteria capable of solubilizing P complexes of aluminum and iron in soils with low levels of available P and their effect on growth of peanut plants.

Materials and methods Collection of rhizospheric soil samples Two hundred fifty rhizospheric soil (30-cm depth) samples collected over five districts of Odisha viz., Balasore, Cuttack, Khordha, Keonjhar, and Mayurbhanj were analyzed for soil reaction (pH) following standard method (Jackson 1967). Isolation and screening of native phosphorous-solubilizing bacteria Enumeration of P-solubilizing bacteria from the collected soil samples was done using National Botanical Research Institute Phosphate (NBRIP) growth medium (Nautiyal 1999) supplemented with tricalcium phosphate (TCP). The diameters of clear zone produced by the isolates were measured using zone scale. NBRIP broth with inorganic phosphates of calcium, aluminum, iron(II), and iron(III) were prepared, and the PSB strains were inoculated. Four replicated broth for each PSB isolates were taken to minimize the error. The broth cultures were incubated at 30 ± 2 °C till 192 h and then centrifuged at 10,000 rpm for 30 min. Water-soluble phosphorus in the supernatant was measured spectrophotometrically at 660 nm by the chloromolybdic acid method as described by Jackson (1967). Isolation of genomic and plasmid DNA Genomic and plasmid DNA were isolated from bacterial cultures grown at 30 ± 0.1 °C following the standard techniques (Jimenez et al. 2011; Jensen et al. 1994). The profiles were visualized through UV transilluminator (312 nm) and photographed using a gel photo documentation system. Pot culture experiment Five bacterial strains were cultured in 100 mL nutrient broth in 250-mL Erlenmeyer flasks at 28 °C and 120 rpm for 72 h to attain optical densities of 0.9 (108 to 109 CFU/ mL) at 620 nm. The broths were then centrifuged at

Contribution of native phosphorous-solubilizing bacteria

12,000 rpm; the pellets obtained were washed thrice with 0.1 M phosphate buffer (pH 7.0) and then dissolved in phosphate buffer (cell count 3.0 × 108 CFU/mL). Peanut (A. hypogaea L. cv. Tag 24) seeds were surface sterilized with 1% NaOCl for 6 min and then repeatedly (six times) rinsed with sterile distilled water for 15–20 min. Sterilized seeds were then placed in glass petri dishes and soaked in phosphate buffer for 2 h (Dey et al. 2004; Fernández et al. 2007). For each seed, 5 mL of phosphate buffer was used. Three inoculated seeds were sown in each of earthen pots (size 10″) filled with 10 kg unsterilized soil (red, sandy loam, pH 5.40, exchangeable Al3+ 0.40 cmol(p+)/kg, organic carbon 4600 mg/kg, available N 69.91 mg/kg, available P 5.24 mg/kg, available K 65.03 mg/kg, Ca-P 42.57 mg/kg, and non-occluded Al and Fe-P 281.50 mg/kg). In each pot, two peanut plants were maintained till maturity, while one peanut plant per pot was uprooted at 45 days after sowing (DAS) for measurement of root length, nodule no., and nodule dry weight. The pot experiment comprised of 12 treatments (Table 5) including one control (N and K only) and replicated thrice in a statistically randomized block design. The fertilizer sources N (10 mg/kg) as urea (CH4N2O) and K (16.602 mg/kg) as muriate of potash (KCl) were applied to all the treatments, while P at 8.728 mg/kg was applied as single superphosphate [Ca(H2PO4)2] following the treatment schedule.

Inorganic P fractions of soils Rhizosphere soil (0–15 cm) from each pot were collected at harvest and analyzed for Ca-P and non-occluded Al and Fe-P following modified Chang and Jackson (1957) method of P fractionation (Olsen and Sommers 1982). Available phosphorous in the soil at harvest was determined by Bray’s 1 method (Bray and Kurtz 1945) as outlined by Page et al. (1982).

Yield attributes of peanut At harvest, the plants were measured for plant height (cm), no. of total pods, pod yield per plant (g), and haulm yield per plant (g). Shoot and kernel P concentrations were measured in spectrophotometer (Page et al. 1982).

Statistical analysis Data were statistically analyzed by the software R (version 3.2.2) and tested with Duncan’s new multiple range test at 5% critical range using the package Bagricolae.^

Results Collection of soil samples and characterization of bacterial isolates GPS-based rhizospheric soil samples (250 nos.) were collected from five districts of Odisha viz., Balasore, Cuttack, Khordha, Keonjhar, and Mayurbhanj, and only 106 nos. with pH ≤ 5.50 were selected for enumeration of phosphatesolubilizing bacteria. From each district, one bacterial isolate with highest P-solubilizing efficiency viz., Balasore BLS18 (31.0 mm), Cuttack CTC12 (31.0 mm), Khordha KHD08 (21.0 mm), Keonjhar KJR03 (20.0 mm), and Mayurbhanj K1 (26.0 mm) exhibiting large clear zones were selected for further study (Table 1 and Fig. 1). Among the five BLS18 and CTC12 were gram +ve rods, while the rest three were gram −ve rods. Comparative P-solubilizing efficiency of the bacterial isolates with Ca, Al, and Fe The five isolates were incubated in NBRIP broth medium supplemented with Ca 3 (PO 4 ) 2 , AlPO 4 , FePO 4 , and Fe3(PO4)2, and the soluble phosphorous (P) and cell density in the incubated broth were measured at 48 and 192 h (Tables 2 and 3). Among the P-supplemented sources, solubilization was maximum in Ca3(PO4)2 followed by FePO4, Fe3(PO4)2, and AlPO4 in declining order. At 48 h, the strain CTC12 recovered maximum soluble P (127.25 mg/L) followed by KJR03 (116.21 mg/L) and KHD08 (115.50 mg/L), respectively, with TCP as P source. With AlPO4 as P source, KHD08 recorded maximum soluble P (8.25 mg/L) followed by CTC12 (5.29 mg/L) and BLS18 (5.29 mg/L). But with FePO4 and Fe3(PO4)2 as P source, CTC12 recovered maximum soluble P (69.25 and 18.00 mg/L) followed by KHD08 (45.83 and 10.35 mg/L). Continuing the incubation further till 192 h, maximum soluble P (465.25 mg/L) was recovered in the broth with TCP by the strain KHD08, while the strains BLS18, KJR03, and K1 accounted for the lowest recovery (340.00 mg/L). Among the five strains, CTC12 exhibited maximum solubilization efficiency with AlPO4 (40.00 mg/ L), FePO4 (175.50 mg/L), and Fe3(PO4)2 (47.40 mg/L) at 192 h. The results further revealed that after 48 h of incubation, CTC12 maintained respective highest cell counts (log CFU/ mL) of 9.09, 8.56, 8.78, and 8.51 in broths with inorganic P sources as Ca3(PO4)2, AlPO4, FePO4, and Fe3(PO4)2, while the strain K1 maintained the lowest cell count (7.85) with AlPO4. However, after 192 h of incubation, CTC12 recorded the highest cell counts (log CFU/mL) of 8.94 and 10.91, respectively, with AlPO4 and FePO4. But with Ca3(PO4)2 and Fe3(PO4)2 as P sources, KHD08 recorded the highest cell count (log CFU/mL) of 11.29 and 9.34, respectively.

M. Pradhan et al. Table 1

Location details of the PSB strains

Districts

Blocks

Village

pH

Isolate code

Diameter of clear zone at 48 h (mm)

Gram’s reaction

Shape

Balasore

Nilagiri

Sarupala

5.39

BLS18

31.0

+ve

Rod

Cuttack Khordha

Athagarh Begunia

Echhapur Balarampur

5.05 5.37

CTC12 KHD08

31.0 21.0

+ve −ve

Rod Rod

Keonjhar

Bansapal

Rangadihi

5.38

KJR03

20.0

−ve

Rod

Mayurbhanj

Muruda

Chitrada

4.92

K1

26.0

−ve

Rod

The pH of the cultured supernatant ranged between 3.33 and 4.89 at 48 h and 3.50 and 4.53 at 192 h irrespective of the strains used. A negative correlation was observed between soluble P and pH of the cultured supernatant at 48 and 192 h of incubation (Figs. 2, 3, 4, 5, 6, 7, 8, and 9). At 192 h, an inverse correlation with respective R2 values of 0.974, 0.867, 0.810, and 0.916 was recorded in mediums supplemented with Ca3(PO4)2, AlPO4, FePO4, and Fe3(PO4)2.

band of genomic DNA (25.784 kbp) followed by B. cepacia K1 (25.526 kbp). The highest no. of plasmid DNA (five nos.) was obtained from B. amyloliquefaciens strain CTC12 which ranges 2.249 to 10.000 kbp. B. cepacia strain KJR03 as well as strain K1 also exhibited two nos. of plasmid DNA.

Molecular characterization

Post harvest rhizospheric soils were characterized for available P, Ca-P, non-occluded Al-P and Fe-P, and exchangeable Al3+ (Table 5). Among the inorganic P fractions, values of nonoccluded Al-P and Fe-P were maximum followed by Ca-P. Post harvest available P of different potted soils ranged from 4.16 to 8.11 mg/kg, while Ca-P and non-occluded Al-P and FeP ranged from 22.55 to 46.29 mg/kg and 142.82 to 281.34 mg/kg, respectively. The highest available P (8.11 mg/kg) was obtained in the soil biofertilized with B. amyloliquefaciens CTC12 + 100% P as SSP, while the uninoculated control pot recorded the lowest (4.16 mg/kg). Among the PSB inoculated pots, B. amyloliquefaciens CTC12 recorded the highest available P (6.88 mg/kg) followed by B. cepacia KHD08 (6.57 mg/kg). B. cereus BLS18 inoculated pots showed the lowest available P (5.89 mg/kg). Pots applied with 100% P as SSP accounted for highest Ca-P (42.66 mg/kg) and non-occluded Al-P and Fe-P (281.34 mg/kg). Inoculation with PSB strains resulted in reduced levels of exchangeable aluminum in rhizosphere soil. The pots treated with P as SSP only recorded the highest concentration of each Al3+ (0.44 cmol(p+)/kg) followed by the uninoculated control pots, but pots inoculated with strain KHD08 recorded lowest concentration of each Al3+ (0.07 cmol(p+)/kg).

Biochemical tests of the five isolates (data not shown) were found to be in agreement with molecular (16S ribosomal RNA (rRNA)) analysis (data not shown). Phylogeny of the isolates BLS18, CTC12, KHD08, KJR03, and K1 figured out to be strains of Bacillus cereus (KT582541), Bacillus amyloliquefaciens (KT633845), Burkholderia cepacia (KT717633), B. cepacia (KT717634), B. cepacia (KM030037), respectively. The genomic and plasmid DNA profiles (Table 4 and Figs. 10 and 11) revealed that the molecular weights of genomic DNAs ranged between 25.784 and 23.947 kbp. B. cepacia strain KJR03 exhibited the heaviest

Effect of PSB on inorganic P fractions and exchangeable Al3+ in peanut rhizosphere

Effect of PSB on root length, nodule no., and nodule dry weight Fig. 1 Halo zone produced by the five PSB strains (BLS18, CTC12, KHD08, KJR03, and K1) in NBRIP agar medium after 48 h of incubation. The largest zones (31 mm) were produced by BLS18 and CTC12, whereas the smallest (20 mm) was with KJR03

Inoculation of peanut with isolates CTC12 and KHD08 in combination with SSP significantly enhanced the root length in the pot culture experiment (Table 6). Pots treated with 100% P as SSP and inoculated with B. cepacia KHD08

Tested by Duncan’s multiple range test with 5% critical range. Means represented by the same letter are not significantly different. Data given in above are average values of four replicates ± standard error of mean (SEM)

19.25 ± 0.675bc 87.35 ± 2.104b 7.07 ± 0.058b 102.80 ± 1.848c K1

2.35 ± 0.042d

37.25 ± 0.443b

340.00 ± 6.565b

22.50 ± 2.870c

42.00 ± 1.568a 36.75 ± 1.724ab 168.75 ± 1.430a 67.75 ± 1.772b 10.35 ± 0.090b 7.80 ± 0.203b 115.50 ± 3.171b 116.21 ± 1.846b KHD08 KJR03

8.25 ± 0.018a 3.75 ± 0.026c

45.83 ± 0.275b 25.55 ± 0.480b

465.25 ± 4.702a 340.00 ± 7.077b

34.50 ± 3.135ab 27.50 ± 1.947bc

15.50 ± 0.749c 47.40 ± 2.398a 135.50 ± 2.434ab 175.50 ± 2.548a 5.29 ± 0.032b 18.00 ± 0.147a 110.40 ± 1.746bc 127.25 ± 2.460a BLS18 CTC12

5.29 ± 0.032b 5.29 ± 0.018b

34.10 ± 0.416b 69.25 ± 0.958a

340.00 ± 7.446b 393.30 ± 5.034ab

30.10 ± 1.923abc 40.00 ± 2.179a

Fe3(PO4)2 Ca3(PO4)2 Fe3(PO4)2 FePO4 AlPO4 Ca3(PO4)2

48-h incubation Isolates

Soluble P (mg/L)

Table 2

Soluble phosphorous recovery by the bacterial isolates with different inorganic P sources

192-h incubation

AlPO4

FePO4

Contribution of native phosphorous-solubilizing bacteria

recorded the maximum root length of 26.67 cm followed by the treatment T10-B. amyloliquefaciens CTC12 + 100% P as SSP. Uninoculated control pots recorded the lowest root length of 15.33 cm. After 45 DAS, one peanut plant from each replicated treatment was uprooted, and nodule no. and nodule dry weight per plant were recorded (Table 6). The highest no. of nodules (112.33) and nodule dry weight (113.25 mg) per plant were recorded in pots receiving B. amyloliquefaciens CTC12 + SSP. The uninoculated control pots recorded the lowest nodule no. (72.67) and dry weight (73.52 mg). Effect of PSB on peanut yield and P concentration Yield attributing traits (plant height, total no. of pods, pod yield, and haulm yield per plant) of peanut plants were recorded at harvest (Table 7). Lowest plant height (42.5 cm), total no. of pods (12.4), pod yield (8.67 g), and haulm yield (41.72 g) were observed in control. B. amyloliquefaciens CTC12 + 100% P as SSP application recorded maximum plant height (62.5 cm), total no. of pods (27.4), pod yield (24.39 g), and haulm yield (63.92 g) per plant closely followed by B. cepacia KHD08. Phosphorous concentration of shoot and kernel (Table 7) revealed significant influence of PSB application. B. amyloliquefaciens CTC12 recorded maximum concentration of shoot P (0.285%) and kernel P (0.573%) when integrated with 100% P as SSP. But B. cepacia KHD08 recorded higher shoot P (0.245%) and kernel P (0.416%) when applied sole. Control pots without P sources recorded the lowest content of P in shoot (0.126%) as well as in kernel (0.206%).

Discussion Acidic soils are characteristically inefficient in providing optimum P nutrition to plants as the inorganic P most often complexed with Al and Fe and very less with Ca. In Odisha, around 70% soils are acidic (Mitra et al. 2002) which possess a bigger problem for the farming community. In addition, about 75% of the inorganic phosphates of applied inorganic fertilizers often get fixed in soil (Podile and Kishore 2006). For maximizing the yields, usually P fertilizers in excess are needed for soil, but these fertilizers are costly and also get immobilized soon after application. Alternatively, there is a scope to isolate P-solubilizing bacteria which could efficiently solubilize inorganic P particularly from Al-P and Fe-P. These efficient PSB strains can be further exploited as biofertilizer. Though the concept of biofertilizers is very old, still in the present investigation, it has been tried to establish the native bacterial strains from acid soils of Odisha as

M. Pradhan et al. Table 3

Bacterial cell count as influenced by different inorganic P sources

Cell count (log CFU/mL) Isolates

48-h incubation

192-h incubation

Ca3(PO4)2

AlPO4

8.97 ± 0.040b

CTC12 9.09 ± 0.016a KHD08 9.08 ± 0.016a

BLS18

KJR03 K1

FePO4

Fe3(PO4)2

Ca3(PO4)2

8.32 ± 0.059a 8.53 ± 0.029bc

8.26 ± 0.091b

11.19 ± 0.036abc 8.61 ± 0.034bc 10.62 ± 0.059b 9.11 ± 0.034b

8.56 ± 0.059a 8.78 ± 0.035a 8.38 ± 0.051a 8.69 ± 0.022ab

8.51 ± 0.071a 11.26 ± 0.054ab 8.40 ± 0.046ab 11.29 ± 0.043a

9.02 ± 0.017ab 8.32 ± 0.040a 8.48 ± 0.046c 8.38 ± 0.065ab 11.11 ± 0.037c 8.98 ± 0.066b 7.85 ± 0.039b 8.63 ± 0.053abc 8.30 ± 0.056b 11.15 ± 0.056bc

AlPO4

FePO4

Fe3(PO4)2

8.94 ± 0.037a 10.91 ± 0.031a 9.26 ± 0.061ab 8.86 ± 0.049ab 10.70 ± 0.054b 9.34 ± 0.049a 8.48 ± 0.077c 8.49 ± 0.056c

10.60 ± 0.039b 9.23 ± 0.062ab 10.62 ± 0.040b 9.15 ± 0.033b

Tested by Duncan’s multiple range test with 5% critical range. Means represented by the same letter are not significantly different. Data given in above are average values of four replicates ± standard error of mean (SEM).

phosphate-solubilizing bacteria and evaluate the efficiency of the strains in solubilizing inorganic phosphate. Five districts of Odisha having soil reaction (pH) below 5.50 were selected for isolation of native PSB strains. These native isolates were analyzed for P-solubilizing efficiency in the NBRIP agar plate by measuring the diameter of the clear zones. Out of the five isolates selected as P solubilizers, two were gram-positive rods and three were gram-negative rods. Earlier workers have also documented many gram positive and negative bacteria involved in mineral P solubilization (Rodríguez and Fraga1999; Lopez et al. 2011). Following the biochemical and 16S rRNA sequencing, the five isolates were identified and submitted to the NCBI gene bank. Since, the NBRIP growth medium with tricalcium phosphate as the insoluble P source could not be considered as the sole protocol for isolation of efficient P solubilizers (Bashan et al. 2013) and as here is the case of acid soils, which predominantly contains inorganic phosphate complexes of AlPO 4, FePO4, and Fe3(PO4)2 (Bashan et al. 2013; Adhya et al. 2015). Hence, the strains were further investigated for P-solubilizing efficiency with P sources viz., AlPO4, FePO4, and Fe3(PO4)2 supplemented in NBRIP broth mediums, and encouraging results were

obtained. After 48 h of incubation, all five strains could solubilize phosphates from complexes of Al and Fe but co mpara t iv ely l ower th an trica lc i um pho spha te. Balamurugan et al. (2010) had also reported variable inorganic P-solubilizing efficiency of PSBs with different insoluble P sources. It was evident here that Ca-P although represents a major fraction of fixed P, not difficult to breakdown (Bashan et al. 2013), in acid soils, the primary fixed phosphates are mainly composed of Al-P and Fe-P, which is an issue of concern. B. amyloliquefaciens CTC12 showed significantly higher solubilization of fixed P from complexes of Ca and Fe than the rest four strains (BLS18, KHD08, KJR03, and K1) at 48 h. B. cepacia KHD08 solubilized significantly higher P in the medium supplemented with AlPO4 over the rest. On the eighth day, more soluble phosphates were recovered compared to the observations made on the second day. No significant differences were observed on the eighth day of incubation among the strains BLS18, CTC12, and KHD08 in the medium supplemented with AlPO4 and FePO4. With P as Fe3(PO4)2, the PSB strains CTC12, KHD08, and KJR03 were found more efficient in solubilizing phosphates on the eighth day of incubation. Similar observations were also made by Park et al. (2010) and Walpola et al. (2012) where they

Fig. 2 Correlation between soluble P and pH of NBRIP broth (Ca3(PO4)2) over 48 h of incubation

Fig. 3 Correlation between available P and pH of NBRIP broth (Ca3(PO4)2) over 192 h of incubation

Contribution of native phosphorous-solubilizing bacteria

Fig. 4 Correlation between available P and pH of NBRIP broth (AlPO4) over 48 h of incubation

Fig. 6 Correlation between available P and pH of NBRIP broth (FePO4) over 48 h of incubation

have noted that microorganisms possess the ability of mineral P solubilization with hard to dissolve complexes like Al-P and Fe-P. The cell density showed an increasing trend on the eighth day in all the culture medium. Medium with Ca-P seemed to favor the growth and multiplication of all the five strains, whereas in Al-P, comparatively least growth was recorded. The cell density in the culture broth was found positively linked to P-solubilizing efficiency. According to Chen et al. (2006), higher metabolic activities in growth medium can be directly proportional to cell density. A significant drop in pH of the culture medium supplemented with different P sources was recorded during the incubation period. After the eighth day of incubation, the pH of the cultured supernatant ranged from 3.50 to 4.53. A strong inverse relationship was found between soluble phosphates and pH of the culture mediums which was in agreement with findings of Alikhani et al. (2007). The major factor being responsible for P solubilization is acidification of the medium because low molecular weight organic acids behave as chelators and form complexes with the cations (Ca, Al, or Fe) which facilitate release of inorganically bound P (Chaiharn and Lumyong 2009; Gulati et al. 2010; Walpola and Yoon 2013). The inverse relationship between these two factors irrespective of the P sources seemed primarily responsible in inorganic P solubilization. Similar findings were also reported before by Chen et al. (2006).

The five potent phosphate-solubilizing bacteria native to acid soils of Odisha solubilized inorganically complexed P in vitro, and further to establish these results in vivo, we conducted a pot culture experiment in unsterilized soil with peanut as the test crop. The treatments differ with P sources, i.e., P fertilizer and P-solubilizing bacteria, while N and K were common to all. Post harvest of peanut, a significant difference on the inorganic fractions of phosphate was recorded among pots with and without PSB inoculation. The predominant inorganic P fraction was nonoccluded Al-P and Fe-P followed by Ca-P. Al and Fe toxicity is a major problem prevalent in acidic soils (Bashan et al. 2013). The peanut pots treated with only P fertilizer (100% SSP) showed significantly higher Ca-P and nonoccluded Al-P and Fe-P than the bioinoculated pots. Moreover, though not significant, higher values of Ca-P and non-occluded Al-P and Fe-P were observed in the pots treated with sole P fertilizer compared to control. Hence, P fertilizer application enhanced all inorganic P fractions (available P, Ca-P, Al-P, and Fe-P). This clearly indicates that water-soluble P fertilizers contributed more to the less labile inorganic P fractions and very less to the labile P fraction, i.e., Bray’s available P. In low pH soils, the soluble P fertilizers applied to crops immediately form complexes with Fe and Al (Goldstein 1986). The variations among the values of inorganic P fractions of soil might be due to the influence of P-solubilizing

Fig. 5 Correlation between available P and pH of NBRIP broth (AlPO4) over 192 h of incubation

Fig. 7 Correlation between available P and pH of NBRIP broth (FePO4) over 192 h of incubation

M. Pradhan et al. Table 4

Genomic and plasmid DNA profiles of the PSB strains

Isolates

Molecular weight (kbp) of genomic NA

No. of plasmids

Molecular weight (kbp) of plasmids

BLS18

24.474

1

2.691

CTC12

23.947

5

10.000, 6.771, 5.433, 2.776, 2.249

KHD08 KJR03

24.211 25.784

1 2

2.776 2.876, 2.423

Fig. 8 Correlation between available P and pH of NBRIP broth (Fe3(PO4)2) over 48 h of incubation

K1

25.526

2

2.747, 2.357

bacteria. Lower amounts of Ca-P, Al-P, and Fe-P were detected in all the PSB-inoculated pots in comparison to uninoculated pots indicating the efficacy of five P solubilizers which were able to utilize the soluble P from the unavailable fractions of soil. However, higher amounts of Ca-P and non-occluded Al and Fe-P were obtained in the pots receiving P-solubilizing bacteria and inorganic P fertilizer combinedly in comparison to pots with PSB which implied that added P fertilizer might have contributed to the inorganic P fractions of soil. The strain B. amyloliquefaciens CTC12 expressed its potency in solubilizing the mineral P from the inorganic fractions either alone or in combination with P fertilizer followed by B. cepacia KHD08. About 26 to 49% P from the less labile fractions (Al-P and Fe-P) were mobilized to the soluble P fraction owing to the solubilizing effect of the five PSB strains. Our findings are in agreement with the results of Turan et al. (2007) and Yousefi et al. (2011) who suggested that application of biological fertilizers could decrease the plant unavailable P fractions in soils making it available for plants. Seed bacterization of these isolates again decreased the exchangeable Al3+ concentration in the rhizospheric soil. In low pH soils, aluminum in the ionic forms prevails and becomes toxic to crops resulting in inhibition in root

elongation and nutrient uptake. These cationic forms of aluminum combine with phosphatic anions in soil; as consequence, the plant available phosphorous easily get immobilized in acid soils (Zheng 2010). Thus, Al toxicity and P deficiency are the major problems in sustainable agriculture in acid soils. PSB inoculation when combined with chemical P fertilization significantly reduced the exchangeable Al 3+ concentration compared to sole P fertilization. Crop rhizosphere is a heterogeneous and dynamic environment that provides various essential nutrients owing to the powerful plant-microbe relationship (Jeffries et al. 2003). The present study revealed that the five PSB isolates (BLS18, CTC12, KHD08, KJR03, and K1) when applied in combination with SSP significantly enhanced root length and nodulation (pink-colored nodule) over the uninoculated control pots. However, the strains CTC12 and KHD08 either in combination with SSP or sole when applied to peanut showed higher root length and nodulation. Dey et al. (2004) showed significant enhancement in root length and nodulation in peanut plant due to seed bacterization in pot culture. The pot culture study with peanut plant carried out with low pH soil has shown significant increase in shoot and kernel P concentration due to application of P fertilizer and PSB. More amount of P was recovered in kernel compared to shoot. P is the main component needed in seed formation, and more amount of P is found in seed than other plant parts (Sagervanshi et al. 2012). All the five strains with or without P fertilizer were able to enhance the P concentration, and the maximum was by B. amyloliquefaciens CTC12 followed by B. cepacia KHD08. Least variation in P uptake was recorded in pots applied either with PSB or SSP. However, these five PSB strains showed potency in vitro. Studies on peanut plants with P-solubilizing bacteria suggested enhanced shoot P as well as soil P content (Anzuay et al. 2015). The five PSB strains also resulted in enhanced growth in peanut plant and yield enhancement. Though the plant growth-promoting aspects of these strains have not been exploited in laboratory conditions, the significant increase in plant height recorded at harvest of plants were found very encouraging. These

Fig. 9 Correlation between available P and pH of NBRIP broth (Fe3(PO4)2) over 192 h of incubation. Figures 2, 3, 4, 5, 6, 7, 8, and 9 represent the negative correlation between soluble P and pH of the cultured supernatant at 48 and 192 h of incubation with various inorganic phosphorous sources [Ca 3 (PO 4 ) 2 , AlPO 4 , FePO 4 , and Fe3(PO4)2]

Contribution of native phosphorous-solubilizing bacteria Fig. 10 Molecular characterization of five PSB strains isolated from acid soils of Odisha. a Genomic DNA profile of five PSB strains (BLS18, CTC12, KHD08, KJR03, and K1) showing the heaviest band of molecular weight 25.784 kbp in strain KJR03. b Plasmid DNA profile of five PSB strains (BLS18, CTC12, KHD08, KJR03, and K1). The highest no. of plasmid DNA was observed in CTC12 ranged between 2.249 and 10.000

PSB isolates when applied sole increased the plant height by 16 to 31% over control. Previous studies suggested that PSB

Fig. 11 Pot culture experiment with peanut at 60 days after sowing (DAS). a Pot cultures showing growth of peanut with PSB2 (Bacillus amyloliquefaciens CTC12) sole and in combination with P fertilizer. b Pot cultures showing growth of peanut with PSB3 (Burkholderia cepacia KHD08) sole and in combination with P fertilizer

inoculation could simultaneously enhance plant P and plant growth (Reyes et al. 2008; Dey et al. 2004).

M. Pradhan et al. Table 5

Effect PSB on soil inorganic P fractions and exchangeable Al3+

Treatments

Available P (mg/kg)

Ca-P (mg/kg)

Non-occluded Al-P and Fe-P (mg/kg)

Exchangeable Al3+ [cmol(p+)/kg]

Control

4.16 ± 0.100f

42.66 ± 0.123a

277.39 ± 0.096a

0.38 ± 0.036ab

100% P as SSP BLS18

4.97 ± 0.043ef 5.89 ± 0.030de

46.29 ± 0.020a 28.40 ± 0.036bc

281.34 ± 0.061a 191.67 ± 0.053bc

0.44 ± 0.017a 0.09 ± 0.020c

CTC12

6.88 ± 0.046bcd

22.55 ± 0.026c

142.82 ± 0.035c

0.08 ± 0.010c

KHD08

6.57 ± 0.026cd

23.42 ± 0.026c

157.17 ± 0.040bc

0.07 ± 0.010c

KJR03 K1

6.13 ± 0.020cde 6.44 ± 0.021cd

26.50 ± 0.010bc 27.87 ± 0.034bc

168.15 ± 0.051bc 189.48 ± 0.026bc

0.10 ± 0.010c 0.10 ± 0.010c

BLS18 + 100% P as SSP

6.86 ± 0.030bcd

33.48 ± 0.020b

197.36 ± 0.216bc

0.15 ± 0.027c

CTC12 + 100% P as SSP KHD08 + 100% P as SSP

8.11 ± 0.026a 8.02 ± 0.026ab

28.67 ± 0.030bc 29.33 ± 0.026bc

162.26 ± 0.079bc 178.18 ± 0.026bc

0.11 ± 1.015c 0.11 ± 1.015c

KJR03 + 100% P as SSP K1 + 100% P as SSP

6.77 ± 0.036cd 7.20 ± 0.026abc

32.54 ± 0.026bc 32.08 ± 0.020b

200.31 ± 0.036bc 205.60 ± 0.062b

0.20 ± 1.015bc 0.23 ± 1.015bc

CV (%)

10.266

13.713

16.076

10.322

Tested by Duncan’s multiple range test with 5% critical range. Means represented by the same letter are not significantly different. Data given in above are average values of three replicates ± standard error of mean (SEM)

To conclude, integrating B. amyloliquefaciens CTC12 and P fertilizer produced significantly higher no. of total pods, pod yield, and haulm yield over sole application of PSB or SSP. Since the soil used in the investigation was acidic in nature with low plant available P and higher concentrations of sparingly available inorganic P, the applied PSB strains could have mobilized P from the native fractions to enhance growth and yield of peanut. Around 46 to 61% increase in pod yield per plant was observed with sole microbial inoculation which further appreciated by 59 to 111% when integrated with P

Table 6 Effect of PSB on root length and nodulation in peanut

f e r t i l i z e r. A l l t h e f i v e s t r a i n s t e s t e d B . c e re u s , B. amyloliquefaciens, B. cepacia, B. cepacia, and B. cepacia resulted in satisfactory growth and yield of peanut as well soil P availability. Soil being a heterogeneous system, it carries the native microflora which often found to be incompetent enough to produce the desired effect on plant by making P available. Thus, isolation and characterization of potent strains from problematic acid soils, mass culturing, and enhancing population in crop rhizosphere could improve soil P status and plant P uptake.

Treatments

Root length per plant (cm)

Nodule no. per plant

Nodule dry wt. per plant (mg)

Control 100% P as SSP BLS18 CTC12 KHD08 KJR03 K1 BLS18 + 100% P as SSP CTC12 + 100% P as SSP KHD08 + 100% P as SSP KJR03 + 100% P as SSP K1 + 100% P as SSP CV (%)

15.33 ± 0.890d 19.67 ± 1.763c 20.33 ± 1.160bc 20.33 ± 1.209bc 21.67 ± 1.000bc 20.33 ± 0.572bc 20.33 ± 1.525bc 22.00 ± 1.857bc 25.67 ± 0.880a 26.67 ± 1.857a 23.33 ± 0.880ab 23.67 ± 1.456abc 9.270

63.33 ± 4.091c 72.67 ± 2.033c 75.33 ± 2.604c 89.33 ± 2.737b 88.67 ± 2.025b 87.67 ± 2.911b 88.33 ± 2.603b 105.67 ± 2.845a 112.33 ± 1.457a 109.33 ± 2.603a 99.67 ± 3.385ab 106.00 ± 2.903a 7.599

65.35 ± 1.015e 73.52 ± 3.147e 76.65 ± 1.786de 88.62 ± 2.802cd 88.50 ± 2.532cd 87.65 ± 3.443cd 87.67 ± 2.867cd 106.18 ± 3.092ab 113.25 ± 2.681a 109.43 ± 4.853ab 99.36 ± 2.884bc 107.55 ± 2.416ab 7.431

Tested by Duncan’s multiple range test with 5% critical range. Means represented by the same letter are not significantly different. Data given in above are average values of three replicates ± standard error of mean (SEM)

Contribution of native phosphorous-solubilizing bacteria Table 7

Effect of PSB on yield attributes and P concentration in peanut

Treatments

Plant height (cm)

Total no. of pods per plant

Pod yield per plant (g)

P concentration in kernel (%)

Haulm yield per plant (g)

P concentration in shoot (%)

Control

42.5 ± 0.866d

12.4 ± 0.400e

8.67 ± 0.321d

0.206 ± 0.003d

41.72 ± 0.797d

0.126 ± 0.003c

100% P as SSP

52.0 ± 1.736bc

20.8 ± 1.600bcd

18.00 ± 0.250bc

0.389 ± 0.003c

52.11 ± 0.200c

0.240 ± 0.003ab

BLS18 CTC12

51.5 ± 4.583c 55.5 ± 3.014abc

18.6 ± 1.114d 21.8 ± 0.600bcd

16.86 ± 0.320c 18.60 ± 0.312bc

0.386 ± 0.003c 0.414 ± 0.006bc

50.35 ± 0.066c 54.95 ± 0.937bc

0.200 ± 0.003b 0.242 ± 0.004ab

KHD08

52.5 ± 3.775bc

21.6 ± 0.400bcd

18.49 ± 0.207bc

0.416 ± 0.004bc

54.65 ± 2.111bc

0.245 ± 0.003ab

KJR03 K1

49.5 ± 1.323c 50.0 ± 1.803c

19.8 ± 0.872cd 18.8 ± 1.000d

16.88 ± 0.482c 15.64 ± 0.225c

0.402 ± 0.005c 0.394 ± 0.006c

50.95 ± 2.353c 50.66 ± 2.674c

0.232 ± 0.003ab 0.238 ± 0.003ab

BLS18 + 100% P as SSP CTC12 + 100% P as SSP

55.5 ± 0.866abc 62.5 ± 0.500a

20.8 ± 0.529bcd 27.4 ± 1.249a

18.38 ± 0.244bc 24.39 ± 0.121a

0.506 ± 0.003ab 0.573 ± 0.005a

53.85 ± 1.732bc 63.92 ± 5.935a

0.253 ± 0.004a 0.285 ± 0.003a

KHD08 + 100% P as SSP

61.0 ± 1.323a

25.8 ± 1.637ab

23.14 ± 0.403ab

0.557 ± 0.002a

61.85 ± 1.790ab

0.280 ± 0.003a

KJR03 + 100% P as SSP K1 + 100% P as SSP

59.5 ± 1.803ab 56.5 ± 1.000abc

24.8 ± 0.400abc 23.4 ± 0.721abcd

20.44 ± 0.057abc 20.10 ± 0.072abc

0.512 ± 0.005ab 0.508 ± 0.003ab

58.15 ± 3.445abc 56.84 ± 3.450abc

0.268 ± 0.002a 0.256 ± 0.004a

CV (%)

7.578

12.722

15.336

12.239

8.388

11.654

Tested by Duncan’s multiple range test with 5% critical range. Means represented by the same letter are not significantly different. Data given in above are average values of three replicates ± standard error of mean (SEM)

Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest.

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