In vitro Phosphate Solubilization by Enterobacter spp. Isolated from Wheat Rhizosphere

Phosphorus is one of the very important minerals required for the proper plant growth. The availability of phosphorous to plants for uptake and utilization is impaired in alkaline and calcareous soil due to the formation of poorly soluble calcium phosphate minerals. Adding fertilizer phosphorous at normal rates and with conventional methods does not result in optimal yield and crop quality in these soils. The use of phosphate solubilizing bacteria can prove to be effective measure to provide phosphorous to the wheat plants to increase the productivity. In the present investigation, a total of 15 isolates were obtained from wheat rhizosphere soil samples. The isolates were subjected to primary and secondary screening and IKas4 and IH37 which showed highest phosphate solubilization during secondary screening were selected for subsequent studies. The condition for in vitro phosphate solubilization by the selected isolates was optimized. The isolate IH37 and the isolate IKas4 showed maximum phosphate solubilization of 0.070 μg and 0.99 μg P/ml respectively. The bacterial isolates were gram negative, non-spore forming rods. On the basis of the 16SrDNA sequencing isolates IKas4 and IH37 were identified as Enterobacter aerogenes and Enterobacter sp. respectively.

Phosphorus is one of 17 nutrients essential for plant growth.Phosphorus is classified as a major nutrient that is frequently required by crops in relatively large amounts.The total P concentration in agricultural crops generally varies from 0.1 to 0.5 percent 1 .In various plant energy reactions P plays a vital role in virtually every plant process that involves energy transfer 2 .P is a vital component of the substances that are building blocks of genes and chromosomes.Large quantities of P are found in seeds and fruit where it is believed essential for seed formation and development.P is also a component of phytin, a major storage form of P in seeds.About 50 percent of the total P in legume seeds and 60 to 70 percent in cereal grains is stored as phytin 3 .Movement of nutrients within the plant depends largely upon transport through cell membranes, which requires energy in the form of ATP and other P compounds to oppose the forces of osmosis 4 .P is taken up mostly as the primary orthophosphate ion (H 2 ), but some is also absorbed as secondary orthophosphate (HPO 4 = ).P may be stored in the root or transported to the upper portions of the plant 5 .
Plants acquire phosphorous from soil solution as phosphate anions which are extremely reactive and get immobilized through precipitation with cations such as Ca 2+, Mg 2+, Fe 3+ and Al 3+ , depending on the particular properties of the soil.Several soil microorganisms known as phosphate solubilizing bacteria (PSB) have the ability to solubilizing insoluble mineral phosphate by producing various organic acids, siderophores, mineral acids, protons, humic substances, CO 2 and H 2 S.This results in acidification of the surrounding soil, releasing soluble orthophosphate ions (H 2 PO 4 -, HPO 4 2-and PO 4 3-) which can be readily taken up by plants 6 .
Almost 75 to 90% of added P fertilizer in agricultural soils is precipitated by iron, aluminum and calcium complexes present in soils.Furthermore, phosphatic fertilizers are expensive, and excessive use of rock phosphate (RP) is potentially and environmentally undesirable 7 .
Wheat takes up P throughout the growing season.Total P uptake by wheat is about 0.5 to 0.6 lb P 2 O 5 /bu (https://www.cropquest.com/2013/09/01/importance-phosphorus-fertilizer-wheat/).Deficiency of P leads to various side effects in the plant growth like reduction in leaf expansion and leaf surface area, as well as the number of leaves.It also leads to a decrease in the shoot root dry weight ratio.The processes of carbohydrate utilization become slow, while carbohydrate production through photosynthesis continues.This results in a buildup of carbohydrates and the development of a dark green leaf color.Since P is readily mobilized in the plant, when a deficiency occurs the P is translocated from older tissues to active meristematic tissues, resulting in foliar deficiency symptoms appearing on the older (lower) portion of the plant.Other effects of P deficiency on plant growth include delayed maturity, reduced quality of forage, fruit, vegetable, and grain crops, and decreased disease resistance 8 .
Alkaline soil is defined as soil with pH greater than neutral, typically 7.5 to 8.5 and calcareous soil is defined as having the presence of significant quantities of free excess lime (calcium or magnesium carbonate).Lime dissolves in neutral to acid pH soil, but does not readily dissolve in alkaline soil and, instead, serves as a sink for surface adsorbed calcium phosphate precipitation 9 .At low pH fixed forms of the P precipitate with calcium ions and at low pH they precipitate with iron and aluminum.
The phosphate solubilizing microbes can be a potential solution to the P deficiency problem.These microbes convert the insoluble and unavailable phosphates into soluble and available form by production of acids, exchange reactions, acidification, chelation etc. 10 A wide range of phosphate solubilizing bacteria (PSBs) have been isolated range of soil samples like from soil of mangroves 11 , saline -alkaline soil 12 , groundnut rhizosphere soil 13 , black pepper rhizosphere soil 14 , saltern 15 , rhizospheres of vegetables like pea, spinach, etc. 16 The effect of phosphate solubilizing bacteria on various plants has been assessed by various researchers.In most of the cases the growth has been enhanced by the PSBs Ramachandran et al. 14 found that the inoculation of PSB alone and PSB + rock phosphate has increased the shoot growth and dry matter production of the black pepper.The control plants on both the potting media have recorded the lowest shoot length.Similarly growth promotion has also been reported in case of pea plants inoculated with Pseudomonas isolate 17 .

Collection of soil samples
Soil samples from wheat rhizosphere were collected from the different areas of Bilaspur district of Himachal Pradesh (India) viz.Harnora, Kasol, Dhar, Bharathu and Kandrour.

Screening of isolates for phosphate solubilization Primary screening
All the suspected colonies were screened for phosphate solubilization on PKV medium.Isolates showing phosphate solubilizing ability were inoculated at the PKV plate and incubated at 37 0 C. Diameter of clearance zone was measured.The phosphate solubilization efficiency (PSE) was calculated as PSE (%) = [Z-C/C] × 100; where, Z = Halo zone diameter, C = Colony diameter 6 .Secondary screening 25ml Pikovskaya's broth medium with Tricalcium phosphate was prepared and sterilized, inoculated with the specific amount of the isolate.Then the inoculated sample was incubated for 48 hours on rotatory shaker at 37 0 C, after incubation culture broth was centrifuged at 10,000 rpm for 5min and colour development done by using the John's method 19 and optical density calculated at 882nm and readings compared with the standard curve to calculate PSE for each isolate.

Characterization of bacterial isolates
Characterization of phosphate solubilizing bacterial strains was performed by morphogical characteristics and biochemical analysis.The microscopic identification was carried out by gram's staining and endospore staining using oil immersion microscope.Morphological and biochemical tests Bergey's Manual of Systemic Bacteriology 20 .For the bacterial isolates showing maximum phosphate solubilization activity, 16SrDNA sequencing was done by Samved Biotech, Ahmedabad (India).

Isolation and screening of phosphate solubilizing bacteria
Wheat rhizosphere soil samples were collected from five different areas of Bilaspur district of Himachal Pradesh that are Dhar,Bharathu,Kandrour,Harnora and Kasol and a total of 15 morphologically distinct colonies were isolated (Table 1).
Till date many works have been done by various researchers, like Vaquez et al. 11 isolated PSBs from mangrove soil and reported that out of all isolates V. proteolyticus was found to be most active.Ramachandran et al. 14 isolated PSBs from rhizospheres of black pepper and Pseudomonas species showed maximum Phosphate solubilization efficiency.Also, Hameeda et al. 21isolated bacterial strains from samples taken from compost and macrofauna.
Malboobi et al. 22 isolated Pantoea agglomerans, Microbacterium laevaniformans and Pseudomonas putida from potato rhizospheres.Similarly, Kannapiran et al. 23   primary screening technique described by Kundu et al. 6 In the primary screening, only four isolates; IKas 4, IH3 7, IKas6 2 and IH4 2 showed halozones whereas all others did not show any zones.On the basis of the halozone and isolate diameter PSE % was calculated and IKas 4 showed maximum PSE% out of four isolates and minimum efficiency % was shown by IKas6 2 .Kumar et al. 25 also observed the halo zone of solubilization up to 20 mm on Pikovskaya agar plates.Kundu et al. 6 also described that 22 isolates exhibited the PSE less than 20% whereas 5 isolates showed PSE in between 50-100%.Pseudomonas sp. was most efficient phosphate solubilizers on Pikovskaya's agar plates with solubilization index 228±6.12at 7th day incubation was shown by Kannapiran et al. 23 Phosphate solubilization efficiency of various isolates in liquid culture was estimated on the basis of colorimetric determination of phosphorus in culture supernatant with ascorbic acid 19 .Phosphate solubilization efficiency was shown by all the 15 isolates during the secondary screening.Maximum efficiency was shown by IKas 4 and IH3 7 i.e. 0.064±0.0006and 0.064±0.0005whereas least PSE value shown by IH3 1. Kundu et al. 6 (2009) demonstrated the P-solubilization in PVK broth, maximum number of bacteria showed P-solubilization < 50 µg P/ml.The isolates were categorized in different classes on the basis of their ability of the P-solubilization such as <50 µg P/ ml, 100-150 µg P/ml, 150-200 µg P/ml, etc.
As we compare the results of primary and secondary screening we find that all the 15 isolates showed PSE in secondary screening whereas only four formed zones of phosphate solubilization.IKas 4 and IH3 7 showed good PSE values both in primary and secondary screening.
In this research all the 15 isolates were subjected for the secondary screening even if they have not shown any halo zones in the primary screening.So, this shows that primary screening is not a very reliable technique for estimation of P solubilization activity.This is in agreement with observation made by several researchers 6,26,27 .Out of the 15 isolates highest phosphate solubilizing efficiency was shown by IH3 7 and IKas 4 and on its basis isolates showing maximum PSE were selected for subsequent studies.

Optimization
The conditions for maximum phosphate solubilization by the selected isolate were standardized by changing various parameters like pH, time of incubation, agitation, carbon source and nitrogen source.

Effect of pH
Both the selected isolates were inoculated in PKV broth with different pH values; 3, 4, 5, 6, 7, 8 & 9 and incubated at 37 0 C for 48 hrs.Maximum PSE was shown by isolate IKas 4 at 3 pH i.e. 0.949±0.003µg P/ml and minimum at pH 8 i.e. 0.011±0.0006µg P/ml.Similarly IH3 7 has shown maximum PSE value at pH 3.0 i.e. 0.107±0.007µg P/ml and least value 0.023±0.003µg P/ml at pH 8 (Fig. 1) According to the values it was observed that with the increase in the pH, the ability of phosphate solubilization by isolates was decreasing whereas the phosphate solubilization increased when pH was decreased.Afzal et al. 16 also observed that Arthrobacter sp., Bacillus sp. and Rhodococcus erythropolis showed maximum phosphate solubilization at pH 3.0.However, Xiang et al. 28 also reported that the best Phosphate solubilization activity was found to be at pH 8.By Zhu et al. 15 it was found that phosphorus solubilization reached the maximum when the pH value of Ca 3 (PO 4 ) 2 containing medium is 7.0.

Effect of Carbon source
Out of the four sets of the PKV broths with different carbon sources, isolates IKas 4 and IH3 7 inoculated in broth with the dextrose as the carbon source gave the maximum PSE value i.e. 0.064±0.0006µg P/ml and 0.064±0.0005µg P/ml respectively whereas minimum value was given by isolates when sucrose was used as carbon source i.e. 0.0027±0.002µg P/ml and 0.040±0.002µg P/ml (Fig. 2).Studies by Maheshwar and Sathiyavani 13 on different carbon sources like glucose, sucrose, lactose, mannitol and sodium acetate on phosphate solubilization revealed that incorporation of glucose followed by lactose increased solubilization of phosphate and enhanced acid production efficiently by phosphate solubilizing bacteria.Nautiyal 29 reported that the carbon source play an important role in the phosphate solubilization and rate of the phosphate solubilization was increased with increasing concentrations of glucose.Similarly, TCP solubilization activity by Pseudomonas lurida was also reported to be maximum with glucose by Kumari & Gupta 30 .

Effect of Nitrogen source
Both the isolates were inoculated in the PKV broths having four different nitrogen sources; ammonium sulphate, ammonium chloride, ammonium nitrate and ammonium oxalate.For the isolate IKas 4 ammonium sulphate proved to be best nitrogen source as PSE value was maximum for it i.e. 0.034±0.0058µg P/ml and minimum value of PSE was given with the ammonium chloride as the nitrogen source.For the isolate IH3 7 also ammonium sulphate was the best nitrogen source with PSE value 0.064±0.0005µg P/ml whereas least PSE was shown when ammonium oxalate was used as the carbon source (Fig. 3).Ammonium sulphate has been found to be a suitable Nitrogen source for various phosphate solubilising bacteria by several workers.Sagervanshi et al. 31 reported that (NH 4 ) 2 SO 4 with Pikovskaya medium showed maximum P solubilization followed by Casein whereas, Urea and Sodium Nitrate gave less activity.Thakker et al. 32 tested many nitrogen sources like ammonium sulphate, ammonium chloride, potassium nitrate, sodium nitrate, and urea for the solubilization of TCP and RP with Enterobacter aerogenes and again reported ammonium sulphate to be most suitable for the purpose.Similar observations were also made by Kumari & Gupta 30 in case of Pseudomonas lurida.

Effect of incubation time and agitation
The selected isolates were inoculated showed the increase in the phosphate solubilization efficiency from second to the eighth day but after that phosphate solubilization by the Penicillium sp.started decreasing.Kannapiran et al. 23 (2011) showed that the Pseudomonas sp.solubilized phosphates from the medium containing tricalcium phosphate and it solubilized a maximum of 1670 mg ml -1 by 10th day of incubation and beyond which no further solubilization was seen.

Effect of all optimized conditions
The isolate IKas 4 was cultured under the optimized condition (pH-3, C sourcedextrose, ammonium sulphate as N source, shaking conditions, and incubation period-96 hrs) and the Phosphorus solubilization activity was assessed.It was observed that the optimized conditions resulted in increase of phosphorus solubilization efficiency of IKas 4 and the final efficiency was recorded to be 0.99±0.0005µg P/ml while initially it was showing phosphate solubilization activity of 0.064±0.00058.Whereas isolate IH3 7 was when cultured under optimized conditions pH-3, C-source-dextrose, ammonium sulphate as N-source, shaking conditions, and incubation period-96 hrs gave a slight enhancement in the PSE 0.070±0.0051µg P/ml from the initial value of 0.064±0.00051.

Characterization of selected isolates
Selected isolates were characterized on the basis of various biochemical and morphological tests.They were also identified on the basis of 16s rDNA sequences.
Isolate IKas 4 colonies were opaque and white in color.According to the gram reaction results IKas 4 was gram negative and rod shaped.Isolate gave negative results for indole and methyl red test whereas positive results were shown for vogues proskauer, citrate utilization and Catalase test.On the other hand, the isolate IH3 7 colonies were white in color and translucent.According to the gram reaction it was rod shaped gram negative bacteria.It also showed negative results for indole, methyl red, citrate utilization and catalase test, whereas positive result was shown for vogues proskauer test.
On the basis of the 16srDNA sequence, the isolates were identified as Enterobacter aerogenes IKaS4 (Genebank Accession Number : KJ921704) and Enterobacter sp.IH37 (Genebank Accession Number : KJ921705).

CONCLUSION
In the present study isolation of Phosphate solubilizing bacteria was carried out from wheat rhizospheres.A total of 15 isolates were obtained from five rhizosphere soil samples.The isolates were subjected to primary and secondary screening and IKas4 and IH3 7 showing highest Phosphate solubilization during secondary screening (0.064±0.00058 µg P/ml and 0.064±0.00051µgP/ml) were selected for subsequent studies.The conditions for in vitro Phosphate Solubilization by the isolates were optimized.The isolates showed maximum Phosphate solubilization under optimized condition with dextrose, ammonium sulphate, pH-3, incubation period 96 hrs with agitation.The bacterial isolates were Gram negative rod, catalase positive, Indole negative, Methyl Red negative, VP positive, and non-spore former.IKas 4 was identified as Enterobacter aerogenes and IH3 7 was identified as Enterobacter sp. by 16s rDNA sequencing.

Table 1 .
24olated Pseudomonas, Bacillus, Vibrio, Micrococcus, Flavobacterium, Corynebacterium, Alcaligenes and Enterobacter from samples collected from different stations of the Thondi coast.Reena et al.24isolated PSBs from rhizospheres of banana plant and identified Bacillus subtilis, Pseudomonas aeruginosa and Micrococcus sp. as major phosphate solubilisers.In the present investigation, the phosphate solubilizing efficiency (Qualitative) of isolates was determined by the plate assay where 15 isolates were spotted on Pikovskaya's agar plate as per the Colony characteristics of phosphate solubilizing bacterial isolates