Users Online: 1278

Home Print this page Email this page Small font sizeDefault font sizeIncrease font size

Home | About us | Editorial board | Search | Ahead of print | Current issue | Archives | Submit article | Instructions | Subscribe | Contacts | Login 
     

   Table of Contents      
ORIGINAL ARTICLE
Year : 2013  |  Volume : 3  |  Issue : 1  |  Page : 29-33

Isolation and characterization of phosphate-solubilizing bacterial species from different crop fields of Salem, Tamil Nadu, India


Department of Biotechnology, Faculty of Engineering, Vinayaka Missions Engineering College, Vinayaka Missions University, Ariyanoor, Tamil Nadu, India

Date of Submission25-Mar-2012
Date of Acceptance18-May-2012
Date of Web Publication6-Feb-2013

Correspondence Address:
Muruhan Sridevi
Department of Biotechnology, Faculty of Engineering, Vinayaka Missions Engineering College, Vinayaka Missions University, Salem, Tamil Nadu
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


Rights and PermissionsRights and Permissions
   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 Sep 21];3:29-33. Available from: http://www.ijnpnd.com/text.asp?2013/3/1/29/106982


   Introduction Top


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. [1] 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. [2] 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 Top


Soil sample

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 [3],[4] 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 [5] 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. [6] 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. [7],[8]

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.[9] 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 [10],[11]




   Result Top


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.

Colony morphology

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.
Table 1: Morphological characters of bacterial species

Click here to view


Microscopic observation

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].

Biochemical characterization

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

Click here to view


RAPD characterization

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

Click here to view
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

Click here to view
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

Click here to view


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

Click here to view



   Discussion Top


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. [12] All the PSB strains isolated from Southern Indian rhizosphere soils showed efficient solubilization of insoluble phosphate as reported earlier. [13] 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, [14],[15] through their hydroxyl and carboxyl groups which chelate the cation bound to phosphate, thereby converting it into soluble forms. [16]

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. [17] 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. [18]

The PSB effect on medicinal plants is gaining scientific scrutiny, as it is evidenced by an increase in the number of publications. [19],[20],[21] 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. [21] 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. [22] 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. [23],[24]

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. [25],[26] 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, [27] 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 [28] 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. [29],[30] Moreover, in several crops, yield and phosphorus uptake has resulted in improving growth by inoculating PSB. [31]


   Conclusion Top


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 Top


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.

 
   References Top

1.Kannaiyan S, Kumar K, Govindarajan K. Biofertilizer technology for rice based cropping system. Scientific pub. (India), Jodhpur 2004. p. 450.  Back to cited text no. 1
    
2.Arpana N, Kumar SD, Prasad TN. Effect of seed inoculation, fertility and irrigation on uptake of major nutrients and soil fertility status after harvest of late sown lentil. J Applied Biol 2002;12:23-6.  Back to cited text no. 2
    
3.Pikovskaya RI. Mobilization of phosphorous in soil in connection with vital activity of some microbial species. Microbiologiya 1948;17:362-70.  Back to cited text no. 3
    
4.SundaraRao WVB, Sinha MK. Phosphate dissolving organisms in the soil and rhizosphere. J Indian J AgricSci 1963;33:272-8.  Back to cited text no. 4
    
5.De Freitas JR, Banerjee MR, Germida JJ. Phosphate-solubilizing rhizobacteria enhance the growth and yield but not phosphorus uptake of canola (Brassica napus L.). Boil Fert Soil 1997;24:358-64.  Back to cited text no. 5
    
6.Anonymous. Manual of Microbiological Methods. McGraw-Hill Book Co. Inc., New York; 1957.  Back to cited text no. 6
    
7.Eckford MO. Thermophilic bacteria in milk. American J Hygiene 1927;7:201-2.  Back to cited text no. 7
    
8.Blazevic DJ, Ederer GM. Principles of biochemical tests in diagnostic microbiology, New York, USA: Willey and Co.;1975. p. 13-45.  Back to cited text no. 8
    
9.Echeverrigaray S, Grazziotion G, Grazziotin F,Agostini G. Random amplified polymorphisms between two South American subspecies of rattlesnakes (Crotalusdurissuscollilineatus and Crotalusdurissusterrificus). Braz Arch Biol Technol 2001;44:313-7.  Back to cited text no. 9
    
10.Sneath PH, Sokal RR. Numerical taxonomy. 1 st ed. San Francisco, USA: W.H. Freeman and Co.;1973. p. 573. ISBN-10: 0716706970,  Back to cited text no. 10
    
11.Ward JH. Hierachical grouping to optimize an objective function. J Am Statist Assoc 1963;58:236-44.  Back to cited text no. 11
    
12.Vazquez P, Holguin G, Puente M, Cortes AE, Bashan Y. Phosphate solubilizing microorganisms associated with the rhizosphere of mangroves in a semi arid coastal lagoon. Biol Fert Soils 2000;30:460-8.  Back to cited text no. 12
    
13.Villegas J, Fortin JA. Phosphorus solubilization and pH changes as a result of the interactions between soil bacteria and arbuscularmycorrhizal fungi on a medium containing NO 3 as nitrogen source. Can J Bot 2002;80:571-6.  Back to cited text no. 13
    
14.Goldstein AH. Recent progress in understanding the molecular genetics and biochemistry of calcium phosphate solubilization by gram negative bacteria. Biol Agric Hort 1995;12:185-93.  Back to cited text no. 14
    
15.Kim KY, Jordan D, McDonald GA. Enterobacteragglomerans, phosphate solubilizing bacteria, and microbial activity in soil: Effect of carbon sources. Soil Biol Biochem 1998;30:995-1003.  Back to cited text no. 15
    
16.Kpomblekou K, Tabatabai MA. Effect of organic acids on release of phosphorus from phosphate rocks. Soil Sci1 994;158:442-53.  Back to cited text no. 16
    
17.Rashid M, Khalil S, Ayub N, Alam S, Latif F. Organic acids production and phosphate solubilization by phosphate solubilizing microorganisms (PSM) under in vitro conditions. Pak J Biol Sci 2004;7:187-96.  Back to cited text no. 17
    
18.Goldstein AH. Involvement of the quinoprotein glucose dehydrogenase in the solubilization of exogenous phosphates by Gram-negative bacteria.In: Torriani-Gorini A, Yagil E, Silver S, editors.Phosphate in microorganisms: Cellular and molecular biology.Washington, DC, USA:ASM Press;1996. p. 197-203.  Back to cited text no. 18
    
19.Earanna N. Response of Stevia rebaudiana to biofertilizers. Karnataka J Agric Sci 2007;20:616-7.  Back to cited text no. 19
    
20.Das K, Dang R, Shivananda TN. Influence of bio-fertilizers on the availability of nutrients (N, P and K) in soil in relation to growth and yield of Stevia rebaudiana grown in South India. Int J Appl Res Nat Prod 2008;1:20-4.  Back to cited text no. 20
    
21.Mamta, Praveen Rahi, Vijaylata Pathaniad, Arvin Gulati, Bikram Singh, Ravinder Kumar Bhanwra, Rupinder Tewari. Stimulatory effect of phosphate-solubilizing bacteria on plant growth, stevioside and rebaudioside: A contents of Stevia rebaudiana Bertoni. Appl Soil Ecol 2010;46:222-9.  Back to cited text no. 21
    
22.Igual JM, Valverde A, Cervantes E, Velazquez E. Phosphate-solubilizing bacteria as inoculants for agriculture: Use of updated molecular techniques in their study. Agronomie 2001;21:561-8.  Back to cited text no. 22
    
23.Aldana L, Mejia EG, Craigmill A, Tsutsumi V, Borunda JA, Panduro A, Rincon AR. Cypermethrin increases apo A-1 and apo B mRNA but not hyperlipidemia in rats. Toxicol Lett 1998;95:31-9.  Back to cited text no. 23
    
24.Nair RR, Abraham MJ, Lalithakunjamma CR, Nair ND, Aravindakshan CM. A pathomorphological study of the sublethal toxicity of cypermethrin in Sprague Dawley rats. Int J Nutr Pharm Neurol Dis 2011;2:179-83.  Back to cited text no. 24
    
25.Franklin RB, Taylor DR, Mills AL. Characterization of microbial communities using randomly amplified polymorphic DNA (RAPD). J Microbiol Methods 1999;35:225-235.  Back to cited text no. 25
[PUBMED]    
26.Uphoff HU, Felske A, Fehr W, Wagner-Dobler I. The microbial diversity in picoplankton enrichment cultures: A molecular screening of marine isolates. FEMS Microbiol Ecol 2001;35:249-58.  Back to cited text no. 26
    
27.Khan M, Zaidi A, Wani P. Role of phosphate-solubilizing microorganisms in sustainable agriculture : A review. Agron Sustain Dev 2006;26:29-43.  Back to cited text no. 27
    
28.Mostafa GG, Abo-Baker AA.Effect of bio and chemical fertilization on growth of sunflower (Helianthus annuus L.) at South valley area. Asian J Crop Sci 2010;2:137-46.  Back to cited text no. 28
    
29.Jilani G, Akram A, Ali RM, Hafeez FY, Shamsi IH, Chaudhry AN, Chaudhry AG. Enhancing crop growth, nutrients availability, economics and beneficial rhizospheremicroflora through organic and biofertilizers. Ann Microbiol 2007;57:177-83.  Back to cited text no. 29
    
30.Yazdani M, Bahmanyar MA, Pirdashti H, Esmaili MA. Effect of Phosphate solubilization microorganisms (PSM) and plant growth promoting rhizobacteria (PGPR) on yield and yield components of Corn (Zea mays L.). Proc World Acad Science Eng Technol 2009;37:90-2.  Back to cited text no. 30
    
31.Hameeda B, Harini G, Rupela OP,Wani SP, Reddy G. Growth Promotion of Maize by Phosphate Solubilizing Bacteria Isolated from Composts and Macrofauna. Microbiol Res 2006;163:234-42.  Back to cited text no. 31
[PUBMED]    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2]



 

Top
 
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
    Abstract
   Introduction
    Materials and Me...
   Result
   Discussion
   Conclusion
   Acknowledgment
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed24562    
    Printed227    
    Emailed1    
    PDF Downloaded1819    
    Comments [Add]    

Recommend this journal