|Year : 2013 | Volume
| Issue : 1 | Page : 29-33
Isolation and characterization of phosphate-solubilizing bacterial species from different crop fields of Salem, Tamil Nadu, India
Alok Ranjan, Mangai Rajan Mahalakshmi, Muruhan Sridevi
Department of Biotechnology, Faculty of Engineering, Vinayaka Missions Engineering College, Vinayaka Missions University, Ariyanoor, Tamil Nadu, India
|Date of Submission||25-Mar-2012|
|Date of Acceptance||18-May-2012|
|Date of Web Publication||6-Feb-2013|
Department of Biotechnology, Faculty of Engineering, Vinayaka Missions Engineering College, Vinayaka Missions University, Salem, Tamil Nadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Phosphate-solubilizing bacterial strains (PSB) were isolated from maize, onion, jasmine, and tomato rhizosphere soils from four different localities of Salem (Tamil Nadu) - (i) Ammapalayam, (ii) Attayampatty, (iii) Seeragapadi, and (iv) Ariyanoor. Twelve efficient PSB isolates were selected from the colonies based on their ability to form clear zone on Pikovskaya's agar medium. The isolated PSB released high amount of phosphorus from calcium phosphate. The efficient bacterial strains isolated from rhizosphere soils released high amount of phosphorus. Oligonucleotide primers in RAPD characterization showed genetic variation among the bacterial strains. Amplified fragments showed 40% shared and 20% unique polymorphic bands. The rest 40% was monomorphic bands.
Keywords: Jasmine, onion, phosphate, phosphate-solubilizing bacteria, rhizosphere, random amplified polymorphic DNA
|How to cite this article:|
Ranjan A, Mahalakshmi MR, Sridevi M. Isolation and characterization of phosphate-solubilizing bacterial species from different crop fields of Salem, Tamil Nadu, India. Int J Nutr Pharmacol Neurol Dis 2013;3:29-33
|How to cite this URL:|
Ranjan A, Mahalakshmi MR, Sridevi M. Isolation and characterization of phosphate-solubilizing bacterial species from different crop fields of Salem, Tamil Nadu, India. Int J Nutr Pharmacol Neurol Dis [serial online] 2013 [cited 2019 Mar 18];3:29-33. Available from: http://www.ijnpnd.com/text.asp?2013/3/1/29/106982
| Introduction|| |
Biofertilizers are the products consisting of selected and beneficial living microbes which are added to soil as microbial inoculants. They are gaining importance because of ecofriendly, non-hazardous, and nontoxic nature. Several organisms such as Cyanobacteria, Azolla, Rhizobium, endophytic diazotrophs, and phosphate-solubilizing microorganisms are presently being used as biofertilizers.  A greater part of soil phosphorus, approximately 95 to 99%, is present in the form of insoluble phosphates and hence cannot be utilized by plants. Great proportion of phosphorous in chemical fertilizer becomes unavailable to the plants after its application in the soil.  This is due to the formation of strong bonds between phosphorous with calcium and magnesium in alkaline pH and the same bonds with iron and aluminum in acidic soils. The mobility of this element is very slow in the soil and cannot respond to rapid uptake by plants. This causes the creation and development of P depleted zones near the area of roots and soil in rhizosphere.
Moreover, the phosphorous deficiency leads to formation of small leaves, weak stem, and slow development.
Plant growth-promoting bacteria (PGPB) are soil and rhizosphere bacteria that can benefit plant growth by different mechanisms, and P-solubilization ability of the microorganisms is considered to be one of the most important traits associated with plant P nutrition. Due to the negative environmental impacts of chemical fertilizers and their increasing costs, the use of PGPB is advantageous in the sustainable agricultural practices.
Phosphate-solubilizing microorganisms play an important role in supplementing phosphorus to the plants, allowing a sustainable use of phosphate fertilizers. Microorganisms are involved in a range of process that affect the transformation of soil Phosphorus (P) and thus are integral component of the soil 'P' cycle. Several mechanisms like lowering of soil pH by acid production, ion chelation, and exchange reactions in the growth environment have been reported to play a role in phosphate solubilization by PSMs. Phosphate-Solubilizing Bacteria (PSB) is slowly emerging as important organisms for the soil improvement. Aim of our present study is to isolate the PSB from maize, tomato, jasmine, and onion crop fields and to study their morphological characteristics.
| Materials and Methods|| |
The soil samples are collected from the fields of maize, onion, jasmine, and tomato rhizosphere from four different zones of Salem, Tamil Nadu, India. Rhizosphere soil was collected in polythene covers and refrigerated.
The agroclimatic zones were used for the sample collection from different places
[(A) MoolaiKarattuKadu, Jagirammapalayam Village, (B) KaduvankaranKadu, Attayampatty, (C) PeriyaSeeragapadi, (D) Ariyanoor] Salem, Tamil Nadu, India.
Homogenization of soil samples
Soil samples were homogenously suspended in double distilled water in the ratio of 1:2 (wet w/v) and centrifuged at 5000 rpm for 5 minutes at 25°C.
Isolation of microorganisms capable of phosphate solubilization
After homogenization, 1 g of each soil samples were serially diluted up to 10 -4 dilution. Then, 1 ml from each solution of dilution 10 -3 and 10 -4 were plated on Pikovskaya's Agar Medium by using Pour Plate Method. Pikovskaya's Agar Medium , is a selective medium for isolating PSB Species. The inoculated plates were incubated aerobically at 32°C for 9 to 10 days.
Screening and selection of phosphate-solubilizing bacteria
After incubation, the colonies showing clear zone of phosphate solubilization were counted  and expressed as colony forming unit (cfu) per gram of soil. Single, well-separated colonies, from each sample, which grew on plates showing clear zones were picked and restreaked onto fresh Pikovskaya's solid medium using quadrant streak method. This procedure was repeated until pure culture with high P solubilization or mineralization was obtained. The strains which showed clear zones were inoculated into nutrient broth and incubated at 28°C ± 2 for 72 h at 100 rpm.
Morphological and biochemical study
The bacterial species form characteristic colonies on Pikovskaya's Agar Media. Morphology of the isolates was studied by Gram staining using kit (K001-1KT, Hi Media) by the standard procedure.  The stained cells were observed under compound microscope. The Gram reaction and cell morphology for efficient PSB strains were recorded. The isolates were examined for starch hydrolyses and catalase. ,
Isolation and quantification of DNA
Bacterial cells from freshly grown cultures on nutrient broth medium were used for DNA isolation processes. DNA was isolated using the phenol/chloroform/isoamyl alcohol method (http://www.bio.vu.nl/geomicrob/protocols/) with slight modifications. The Genomic DNA isolated from bacterial strains is quantified by Agarose gel electrophoresis.
Standardization of protocol for RAPD analysis
For fingerprinting and diversity analysis, PCR (Polymerase Chain Reaction) amplification conditions were optimized based on the protocol outlined by Echeverrigaray et al. with minor modifications. In order to obtain high amplification rate and reproducible banding pattern, different duration for hot start, denaturation, and primer annealing, and primer extension were tried.
Random Amplified Polymorphic DNA (RAPD) primers with sequences chosen for analysis ,
| Result|| |
Isolation of phosphate-solubilizing bacteria and their ability to solubilize calcium phosphate
From the rhizosphere soils (Tomato, Jasmine, Onion, and Maize), the total of 12 bacterial species (of 32 samples) were found enriched on Pikovskaya's agar plates. The bacterial species were isolated. After 2-3 days, the efficient bacterial species capable of solubilizing phosphate and formation of phosphate-solubilizing zone (PSZ) are further inoculated on Pikovskaya's agar medium and incubated at 30°C in rotary shaker (100 rpm).
The isolates were able to show PSZ ranging from 6 to 10-mm diameter due to solubilization of calcium phosphate when incubated for 9 to 10 days at 32°C.
Morphologically, all the bacterial isolates exhibited typical bacterial colonization characters. Bacterial species produced white color colonies and some were yellow color colonies. Most of the isolates formed well-developed colonies on Pikovskaya's agar medium within 9 to 10 days of incubation [Table 1]. Three bacterial species isolated were from jasmine, four isolates from maize, three isolates from onion, and two isolates from tomato fields.
Bacterial species were further examined for their Gram's reaction and shape. Characteristically, all the isolates were Gram negative and of rod and round shape [Table 1].
All isolates were found positive to starch hydrolysis test and negative for catalase test.
Quantification of genomic DNA
The genomic DNA was quantified by comparing the band intensity of different isolated DNA. The entire isolated DNA was in high intensity and high concentration [Figure 1].
|Figure 1: Quantification of Isolated Bacterial Genomic DNA of bacterial Isolates by Agarose Gel Electrophoresis|
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A total of 15 bands produced from the selected 4 primers were used for fingerprinting and estimation of genetic diversity among 12 isolates of bacterial strains. For the purpose of illustration, the RAPD fingerprints or electrophoregram generated for 12 bacterial isolates using four primers are presented in Plates [Figure 2], [Figure 3], [Figure 4].
|Figure 2: RAPD Gel profile of bacterial isolates generated using 10-mer random primer no. 1 and primer no. 2. Lane 1-4 and 5-8 are bacterial isolates of Maize and Jasmine Rhizosphere soil samples|
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|Figure 3: RAPD Gel profile of bacterial isolates generated using 10-mer random primer no. 3. Lane 1, 2, 3, 4, and 5 are bacterial isolates of Onion Rhizosphere soil samples|
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|Figure 4: RAPD Gel profile of bacterial isolates generated using 10-mer random primer no. 4. Lane 1 and 2 are bacterial isolates of Tomato Rhizosphere soil samples|
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The number of bands scored for each primer varied from 1 to 9 with an average of 1 band each primer. Of 15 amplification bands, three bands were unique, six bands were shared polymorphic, and six bands were monomorphic, which were informative in revealing the relationship among genotypes [Table 2].
|Table 2: Oligonucleotide primers that showed genetic variation among the bacterial strains|
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| Discussion|| |
Phosphorous is one of the major essential nutrients required for the plant growth. High proportion of phosphate-solubilizing microbes is concentrated in the rhizosphere, and they are metabolically more active than other sources.  All the PSB strains isolated from Southern Indian rhizosphere soils showed efficient solubilization of insoluble phosphate as reported earlier.  Phosphate-solubilizing microorganisms not only provide phosphorous to the plant but also at the same time provide growth-promoting substances like hormones, vitamins, and amino acids. Microbial solubilization of phosphate in soil is correlated with the ability of microbes in producing selected organic acids and extracellular polysaccharides. It is generally accepted that the mechanism of mineral phosphate solubilization by PSB strains is associated with the release of low molecular weight organic acids, , through their hydroxyl and carboxyl groups which chelate the cation bound to phosphate, thereby converting it into soluble forms. 
From the results of morphological study, it is being reaffirmed that the phosphate solubilization by different PSBs (Gram-negative) involved may be with the production of organic acids.  Moreover, it has been reported that Gram-negative bacteria mobilize insoluble phosphate very efficiently, by producing gluconic acid during the extracellular oxidation of glucose catalyzed by quinoprotein glucose dehydrogenase. 
The PSB effect on medicinal plants is gaining scientific scrutiny, as it is evidenced by an increase in the number of publications. ,, The inoculation of PSB significantly increases the plant growth (shoot length, root length, leaf dry weight, stem dry weight, and biomass), available P content in soil as well as its uptake.  The organisms predominantly reported were Enterobacter aerogenes, Micrococcus sp., Pseudomonas aeruginosa, and Bacillus sp. Among the soil bacterial communities, ecto-rhizospheric strains from Pseudomonas and Bacilli, and endosymbiotic rhizobia have been described as effective phosphate solubilizers.  Moreover, use of chemicals in agriculture and livestock production has created potential health hazards, not only to livestock and wildlife, but also to living organisms. ,
To differentiate closely related strains of bacteria, RAPD analysis is commonly used and recent works applied RAPD technique to evaluate differences among total bacterial aquatic communities. , Oligonucleotide primers in RAPD characterization showed genetic variation among the Bacterial strains. Amplified fragments showed 40% shared and 20% unique polymorphic bands. The rest 40% was monomorphic bands. Microorganisms from jasmine does not show any unique polymorphic bands, whereas maize, onion, and tomato showed shared polymorphic bands. Furthermore, onion showed no monomorphic bands.
As PSB isolates comprise a diverse of unrelated bacteria,  further studies are warranted for identification by nucleotide sequencing of 16S rRNA genes and sequence comparison with available data in the GenBank.
The natural subtropical soil supports a diverse group of potential PSB. These P-solubilizing soil bacteria could serve as efficient biofertilizer candidates  for improving the P-nutrition of crop plants and helps to minimize the P-fertilizer application, reduces environmental pollution, and promotes sustainable agriculture. It has been reported that the PSB and plant growth-promoting rhizobacteria together could reduce P fertilizer application by 50% without any significant reduction of crop yield. , Moreover, in several crops, yield and phosphorus uptake has resulted in improving growth by inoculating PSB. 
| Conclusion|| |
From our study, we conclude that the phosphate-solubilizing Gram-negative bacteria were present in maize, corn, jasmine, and onion fields and enrich soil soluble phosphate. More studies are warranted to identify and understand the significance and mechanism underlying the formation of soluble phosphate by PSB and its benefits as bio-inoculants. Further studies in genetic manipulation of PSB to improve their ability to improve plant growth are needed.
| Acknowledgment|| |
The authors thank Dr. K. M. Harinikumar, PhD., Associate Professor, Department of plant biotechnology, University of Agricultural Sciences, Bengaluru, Karnataka, India, for his support in the work.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]