Chapter 7

Microbial DNA extraction

from soil (culture

independent) and its PCR

amplification by different

methods

7.1 Introduction

The diversity of microorganisms in soil is critical to the maintenance of good soil health, because

microorganisms are involved in many important functions such as soil formation, toxin removal,

and elemental cycles of C, N, P, and others. Previously, studies on the development of “bacterial

communities required the isolation of the organisms from sample, followed by a series of

morphological and biochemical tests to identify the bacteria. However, the majorities of bacteria

in the environment are not readily cultivable” (Moyer et al.1994, Marchesi et al. 1998).

“Furthermore, culture dependent community structure analysis produce spatial and heavily

biased results. Since in the mid 1980s, the use of 16S ribosomal nucleic acid–(rRNA) techniques

has facilitated the molecular identification of a wide variety of as yet uncultivated

microorganisms and novel microbial groups in various environments”( Torsvik et al. 1990 ).

There are two approaches for the extraction of DNA from soil: the bacterial fractionation

approach, involves the separation of bacterial cells from the bulk of the soil prior to cell lysis and

recovery of bacterial community DNA, and the direct lysis approach, bacterial cells are lysed

directly in the presence of the soil matrix. Direct lysis methods are more often used than cell

extraction ones because they are less time consuming and give better recovery, resulting in an

extracted DNA more representative of the whole microbial community present in the sample.

“The major disadvantage of direct lysis is that the method also extracts other organic soil

components, such as humic and fluvic acids. Since these contaminants can prevent subsequent

molecular analysis, such as restriction digestion, Polymerase Chain Reaction (PCR), and

cloning” (Olson et al., 1991), an extensive purification steps were then required to successfully

amplify a PCR product, including CsCl-ethidium bromide density gradient centrifugation or the

use of commercial reagents. These steps increase both the complexity and the cost of the

technique. This paper describes in detail a method for extracting DNA from soil which involves

minimal purification prior to PCR amplification. An additional advantage is that only 1 gm of

soil sample is required results in analyzing a soil sample in a short span of time. Thus a PCR

product was obtained rapidly and inexpensively from small amount of soil.

Several methods for extracting microbial DNA from soil have been described. “Many of

these techniques employ extensive purification steps to ensure that the DNA is suitable for use in

the polymerase chain reaction” (Roose-Amsaleg, et al, 2001). Thus, selection of an appropriate

DNA extraction and purification procedure from among the procedures that have been described

to date remains a major problem in the application of molecular techniques to studies of soil and

sediment microbial communities. The objective of this study is to evaluate different methods for

extracting high yield of high-purity microbial community DNA using single soil samples

emphasized PCR amplification in evaluating DNA purity because Taq polymerase is sensitive to

humic contaminants. Amplification of DNA from soil is often inhibited by co-purified

contaminants. A rapid, inexpensive, large-scale DNA extraction methods involving minimal

purification have been developed that is applicable to soil. DNA is also suitable for PCR

amplification using various DNA targets. DNA was extracted from 1g of soil using direct lysis

with liquid nitrogen and SDS followed by sodium acetate precipitation, polyethylene glycol

precipitation, phenol extraction and isopropanol precipitation. This method was compared to

other DNA extraction methods with regard to DNA purity and size.

7.2 Material and methods

The soil sample was collected from Kukrail Forest. Soil samples were immediately placed on dry

ice, mixed, and then stored at -20°C prior to DNA extraction. Subsample (approximately 1 g) of

soil was set aside for use in microbial analysis.

Six DNA extraction methods were evaluated in this study using single type of soil. Three

methods were Mannitol-based DNA extraction method grinded with liquid nitrogen and others

were based on PEG-NaCl method without liquid nitrogen, bead beating without liquid nitrogen

and Sodium phosphate -SDS based method.

7.2.1 DNA extraction using mannitol method with liquid nitrogen

Three different protocols are used in this method which gives a very good yield of DNA

directly isolated from soil sample. Take 1:10 ratio of extraction buffer with respect to soil sample

i.e. 10 ml of isolation buffer is added in 1g of soil sample in all three protocols of this method.

7.2.1.1 Protocol I: Using PEG/NaCl with mannitol

DNA was extracted from soil samples by directly lysing the cells with liq.N2.Soil sample of 1 gm

were mixed with 10 ml of DNA extraction buffer (1M Tris-HCL [pH 8.0], 5 M NaCl, 0.5 M

EDTA (pH-7.8), 10 % CTAB , 20 % SDS and rest of volume Mannitol ) in centrifuge tubes and

incubated for 1 hr at 65°C with occasional stirring. The supernatant were collected after

centrifugation at 1000 rpm for 15 min at room temperature and mixed with half volume PEG and

1 volume of NaCl and incubated at 4 C for overnight. The pellet was recovered by

centrifugation at 12,000 rpm and dissolved in 1ml of TE buffer (Tris-HCl 10 mM, Na2EDTA 1

mM; pH 8.0). The DNA sample was then extracted with PCI; aqueous layer was taken in fresh

tube and allowed to precipitate at 4 C with 1/10th volume of 3 M sodium acetate (pH 5.2) and 2

volumes ethanol. The pellet was redissolved in 50 μl TE buffer.

7.2.1.2 Protocol II: Using Phenol/Chloroform/Isoamylalcohol (P/C/I), with mannitol

DNA was extracted from soil samples by directly lysing the cells with liq.n2.Soil sample of 1 gm

were mixed with 10 ml of DNA extraction buffer (1M Tris-HCL [pH 8.0], 5 M NaCl, 0.5 M

EDTA (pH-7.8), 10 % CTAB , 20 % SDS and rest of volume Mannitol) in centrifuge tubes and

incubated for 1 hr at 65° C with occasional stirring. After centrifugation, the supernatant was

extracted with an equal volume of PCI followed by centrifugation at 12,000 rpm at 4 C.

Aqueous layer was taken and allowed to precipitate overnight at 4 C with 1/10th

volume of 3 M

sodium acetate (pH 5.2) and 2 volumes ethanol. The pellet was recovered by centrifugation at

12,000 rpm and dissolved in 50 μl TE buffer.

7.2.1.3 Protocol III: Using CTAB with mannitol

DNA was extracted from soil samples by directly lysing the cells with liq.n2.Soil sample of 1 gm

were mixed with 10 ml of DNA extraction buffer (1M Tris-HCL [pH 8.0], 5 M NaCl, 0.5 M

EDTA (pH-7.8), 10 % CTAB , 20 % SDS and rest of volume Mannitol) in centrifuge tubes and

incubated for 1 hr at 65° C with occasional stirring. After centrifugation, 50 µl of 5 M NaCl and

50 µl of 10 % CTAB (Cetrimide) (made in 0.7 M NaCl solution CTAB were added to the

supernatant and incubated at room temperature for 10 min. This was followed by addition of

equal volume of PCI and centrifugation at 12,000 rpm 4 C. Aqueous layer was taken and

allowed to precipitate overnight at 4 C with 1/10th

volume of 3 M sodium acetate (pH 5.2) and

2 volumes ethanol. The pellet was recovered by centrifugation at 12,000 rpm and dissolved in 50

μl TE buffer.

7.2.2 DNA extraction using PEG / NaCl method without liquid nitrogen

DNA was extracted from soil samples by directly lysing the cells with liq.N2 .Soil sample of 1

gm were mixed with 10 ml of DNA extraction buffer (120 mM Na2HPO4 (pH 6.8), 5 % SDS

(w/v) and 0.02 g PVPP) in centrifuge tubes and incubated for 1 hr at 65°C with occasional

stirring. The supernatant were collected after centrifugation at 1000 rpm for 15 min at room

temperature and mixed with half volume PEG and 1 volume of NaCl and incubated at 4 C for

overnight. The pellet was recovered by centrifugation at 12,000 rpm and dissolved in 1ml of TE

buffer (Tris-HCl 10 mM, Na2EDTA 1 mM; pH 8.0). The DNA sample was then extracted with

PCI; aqueous layer was taken in fresh tube and allowed to precipitate at 4 C with 1/10th volume

of 3 M sodium acetate (pH 5.2) and 2 volumes ethanol. The pellet was redissolved in 50 μl TE

buffer.

7.2.3 DNA extraction using bead beating without liquid nitrogen

Extraction buffer (120 mM K2HPO4 pH 8.0, 5 % CTAB in 120 mM K2HPO4 pH 8.0 CTAB,

0.50 ml PCI) was mixed with 1g (wet weight) of soil. Glass beads 0.5g, were added and the

sample blended in a Bead-Beater for 2 minutes with intermittent cooling on ice and centrifuge 15

min at maximum speed (10,000 rpm). The sample was incubated at 65° C for 1 h, transferred to

centrifuge tubes and centrifuged at 8000 rpm for 15 min. The supernatant was collected, and the

soil pellet re-extracted with further extraction buffer (10 ml), incubation at 65º C for 10 min and

centrifugation as above. Supernatants were transferred to centrifuge tubes containing 1 volume

of Chloroform and isoamyl alcohol and vortexed. The supernatant was collected by centrifuge

for 10 min at 12000 rpm 0.1 volume 3 M NaAc and 1 volume ethanol (cool), let the DNA to

precipitate for 2 h on ice. Centrifuge 15 min, 12,000 rpm and wash the pellet with 70 % ethanol,

Add 50 µL TE buffer into the DNA pellet.

7.2.4 DNA extraction using sodium phosphate buffer & SDS method with bead beating &

liquid nitrogen

Soil sample of 1 g was introduced into a 25 mL erlenmeyer flask containing 10 ml sodium

phosphate buffer (and 0.1 % Tween 80, pH 7). The mixture was incubated under constant

agitation overnight at room temperature. The supernatant from this suspension was centrifuged at

5000 rpm for 10 min. The pellet was washed four times with TE buffer 50/50 ( 50 mM Tris-HCl,

50 mM EDTA, pH 8 ) at 3000 rpm for 3 min and the cells were lysed mechanically by

maceration of the liquid nitrogen-frozen pellet. The macerate was transferred to a 15-mL

centrifuge tube to which 2 mL TE buffer 50/50 and an equivalent volume of phenol-chloroform-

isoamyl alcohol (25:24:1) were added, and the mixture was centrifuged at 5000 rpm for 10 min.

DNA from the collected supernatant was precipitated by adding 0.7 vol. ice-cold isopropanol and

10 vol. 3 M sodium acetate and chilling this mixture for 2 h at -20°C.Another centrifugation at

5000 rpm for 10 min yielded a DNA pellet, which was washed two times with ice-cold 70 %

ethanol and once with 95 % ethanol. The DNA was resuspended in 50 µL TE buffer (10 mM

Tris-HCl, 0.1 mM EDTA, pH 8.0).

7.2.5 Determination of purity and yield of DNA

The concentration of the DNA in the sample can be measured by monitoring the absorbance of a

dilute solution of the sample at 260 nm, and can be calculated based on the value of 1.0 A260 unit

= 50 μg/ml of DNA, taking into account the dilution factor of the sample (Sambrook et al.,

1989). Purity of the DNA was determined by taking absorbance reading at 230, 260 and 280 nm.

A260 /A280 and A260 /A230 ratios were calculated to evaluate levels of protein and humic acid

impurities respectively in both extraction methods.

7.2.6 PCR amplification of soil extracted DNA

Soil DNA was amplified by PCR (BIORAD). Each 25 l PCR mixture contained 1 l template

DNA, 2.5 l 1x PCR buffer, 1 l of each deoxyribonucleoside triphosphate (dNTP), 1 l of

forward and reverse primers and 0.5 l Taq DNA polymerase The primers used to amplify SSU

ribosomal genes (FP1) 5’-TGGGGAGCAAACAGGATTAG-3’ and (RP1) 5’-

TAAGGTTCTTCGCTTGCTT-3’.The amplification cycle consisted of an initial denaturation

step of 5 min at 94 C, followed by 35 cycles of 1 min at 94 C (denaturation), 1 min at 59 C

(annealing) and 2 min at 72 C (extension), with a final extension step for 10 min at 72 C

Successful amplification was confirmed by agarose gel electrophoresis and ethidium bromide

staining. An aliquot (6 μl) of each amplification reaction was analysed on 0,8 % w/v agarose gels

cast and run in TAE buffer (pH 8.2) .Gels were stained with ethidium bromide and photographed

using transmitted U.V. light using BIO RAD Gel DocTM XR. A 100 base pair marker was

included on gel.

7.3 Results

DNA extraction from soil has three requirements; extraction of high molecular weight DNA;

extraction of DNA free from inhibitors for subsequent molecular biological manipulations to be

performed; representative lysis of microorganisms. It is important to select an extraction and

purification method, which yields DNA of suitable quantity and purity. The extraction methods

are strongly influenced by several parameters, such as incomplete cell lysis, DNA sorption to soil

surfaces, extraction of humic contaminants, and DNA degradation. In this chapter, we tested a

number of DNA extraction methods for their ability to fulfill these requirements. Here six

methods are considered for isolation of DNA and compared. The inclusion of Mannitol in the

extraction buffer has been proved to increase the efficiency of extraction. Further purification of

DNA has been tried with three different procedures, viz., by using PEG-NaCl, CTAB and PCI.

The size of extracted DNA ranged from less than 500 bp to greater than 20 kb in size. Higher

molecular weight DNA is desirable for PCR since the greater the size of the DNA, the less likely

is the formation of chimeras during PCR, hence better for microbial diversity analysis. .

7.3.1 Metagenomic DNA from soil

Various DNA isolation protocols are compared on the basis of DNA yield (Table 7.1). DNA

yield was increased by using liquid nitrogen and bead beating. It was further improved by adding

mannitol in the extraction buffer. The SDS method has been the most widely used cell lysis

treatment from DNA extraction from pure cultures, soils and sediments. Trevors et al., (1992)

found that the SDS-based cell lysis protocol gave the highest DNA yields in comparison with

freezing-thawing lysis protocols. More et al., (1994) showed that the mechanical treatment was

more effective and less selective than chemical lysis, attributed to increased cell lysis. According

to Frostegard et al., (1999) grinding homogenizes the soil and increase the release of bacteria

from inner compartment, making them available for subsequent lysis.

1 2 3 4 5 6 7

Figure 7.1 Visualization of soil DNA showing smear by respective methods. lane 1 Marker;

lane 2 Mannitol -PEG/NaCl method; lane 3- Mannitol P/C/I; lane 4 Mannitol - CTAB; lane

5-DNA extraction using PEG / NaCl Method without Liquid Nitrogen; lane 6 -DNA

extraction using Sodium Phosphate buffer & SDS method with bead beating & Liquid

Nitrogen; and lane 7 -DNA extraction using bead beating without liq. N2.

7.3.2 Determination of purity and yield of DNA

Table 7.1: Amount of DNA extracted from various isolation protocols

DNA Extraction Protocol Amount

(mg/ml)

DNA extraction by PEG / NaCl without liq. N2 0.75

DNA extraction using Phosphate buffer with bead beating without liq. N2

0.80

DNA extraction using Na2HPO4,buffer & SDS method with bead beating &

liq N2

1.25

DNA extraction by Mannitol method with Liq N2

Protocol I: 2.25

Protocol II: 2.50

Protocol III: 2.75

Since organic matter is the major source of inhibitors that may be co-extracted from soil

with the microbial DNA. In particular, humic acids pose a considerable problem and will

interfere in enzymatic manipulations of DNA. The humic materials in soil have similar size and

charge characteristics to DNA resulting in their co-purification, evident by the extractions being

brown in colour. Humic contaminants also interfere in DNA quantitation since they exhibit

absorbance at both 230 nm and at 260 nm, the later used to quantitate DNA. This characteristic

can be used to determine the level of contamination of humic material by examining absorbance

ratios. A high 260/230 ratio (>2) is indicative of pure DNA, while a low ratio is indicative of

humic acid contamination and a high 260/280 ratio (>1.7) is indicative of pure DNA, while a low

ratio is indicative of protein contamination.

Table 7.2 Comparison of DNA extraction methods for humic acid contamination

DNA Extraction Protocol A 260/230 A 260/280

DNA extraction by PEG / NaCl without liq. N2 1.10 1.25

DNA extraction using Phosphate buffer with bead beating

without liq. N2

1.20 1.42

DNA extraction using Na2HPO4,buffer & SDS method with

bead beating & liq N2

1.24 1.69

DNA extraction by Mannitol method with liq N2

Protocol I: 1.89 1.80

Protocol II: 1.94 1.85

Protocol III: 2.05 1.85

When the DNA extraction methods were compared (Table 7.2), the bead beating method and

PEG / NaCl Method with Liquid Nitrogen method consistently extracted DNA with higher

260/230 and 260/280 ratios. This indicated that the DNA was contaminated with fewer humic

acid-like compounds. Furthur more Mannitol based methods and the Sodium phosphate-SDS

method may remove humic acid material very effectively than the other methods of extraction.

7.3.3 PCR Amplification using 16S rRNA primers

The impurities mainly removed by method using Mannitol and SDS, which proved to be

an efficient protocol for extraction. Among the three purification methods that were applied to

soil-extracted DNA, protocols #1, #2, #3 and #6 yielded DNA ready for efficient PCR

amplification. The size of amplicons is estimated 225 bp. Since PCR involves successive

enzymatic reactions and since DNA polymerase requires contamination-free sites. It appears that

extraction method of all three Mannitol Methods (P/C/I, PEG/NaCl and CTAB) and sodium

phosphate SDS method gave the very good quantity of DNA for the amplification by PCR

while other method were not sufficient for the PCR due to probably due to contaminating (DNA-

adhering) substances like humic acid. These contaminating materials were removed and this

could have led to inhibition of the initial PCR product on the original DNA. Humic acid

impurities may affect DNA hybridization efficiency too. In the method using Mannitol with liq.

N2 showed a very good chemical lysis than the other methods so according to this concept

Mannitol may also play a important role in chemical lysis of the cells with the other chemicals

like CTAB, SDS, EDTA and Tris-HCL etc so that the lysis of cells may be good in comparison

to other methods and the yield of soil DNA become high.

1 2 3 4 5 6 7

Figure 7.2 Visualization of PCR Amplification of soil DNA extracted by different methods

showing bands on elecrtophoresis lane 1 Marker; lane 2 Mannitol- PEG/NaCl method; lane

3- Mannitol -P/C/I; lane 4 Mannitol - CTAB; lane 5-DNA extraction using PEG / NaCl

225bp

method without Liquid Nitrogen; lane 6-DNA extraction using bead beating without liq.

N2; lane 7-DNA extraction using Sodium Phosphate buffer & SDS method with bead

beating & Liquid Nitrogen

Thus, we compared the obtained yield of soil DNA by all used methods. Amplification of

DNA by PCR was used to determine its quality. Among the methods only Sodium phosphate-

SDS with liquid nitrogen, all three mannitol Methods with liquid nitrogen, ready for

efficient PCR amplification. And other Bead beating without liq N2, and PEG-NaCl without liq.

N2, even though giving an acceptable level of DNA, was not suitable for PCR amplification.

PCR involves successive enzymatic reactions and since DNA polymerase requires

contamination-free sites. The described method allows the use of large scale preparations

providing greater probability of detecting genes present in low abundance in the soil

environment. Because this method is applicable to even the more challenging heavily

contaminated soils, molecular microbial biodiversity assessment can now be more readily

applied.

7.4 Conclusion

For Metagenomic studies it is important to select an extraction and purification method,

which yields DNA of suitable quantity and purity from the soil. The extraction methods are

strongly influenced by several parameters, such as incomplete cell lysis, DNA sorption to soil

surfaces, extraction of humic contaminants, and DNA degradation. In this study, we tested a

number of DNA extraction methods for their ability to fulfill these requirements. Here six

methods are considered for isolation of DNA and compared. The inclusion of Mannitol in the

extraction buffer has been proved to increase the efficiency of extraction. Further purification of

DNA has been tried with three different procedures, viz., by using PEG-NaCl, CTAB and PCI.

The size of extracted DNA ranged from less than 500 bp to greater than 2 kb in size. Higher

molecular weight DNA is desirable for PCR since the greater the size of the DNA, the less likely

is the formation of chimeras during PCR, hence better for microbial diversity analysis.

Bibliography

1. ABD ALLA, (1994), Phosphatases and the utilization of organic phosphorous by

Rhizobium leguminosarum biovar viceae, Lett. Appl. Microbial, 18, 294-296.

2. ACHAL V, SAVANT V.V, SUDHAKARA, REDDY M, (2007), Phosphate

solubilization by wide type strain and UV-induced mutants of Aspergillus

tubingensis, Soil Biology and Biochemistry, Vol. 39, No.2, pp. 695-699.

3. AHMAD F, AHMAD I, KHAN MS, (2005), Indole acetic acid

production by the indigenous isolates of Azotobacter and Flourescent

pseudomonas in the presence and absence of tryptophan , Turk J. Biol.

29, 29– 34.

4. AHMAD N, JHA KK, (1968), Solubilization of rock phosphate by microorganisms

isolated from Bihar soil, Journal of General and Applied Microbiology, 14, 89–95.

5. ALAVANJA M, SAMANIC C, DOSEMECI M, LUBIN J, TARONE R, LYNCH C,

KNOTT C, THOMAS, K, HOPPIN J, BARKER J, COBLE J, SANDLER D, BLAIR

A, (2003), Use of agricultural pesticides and prostate cancer risk in the

Agricultural Health Study cohort, Am J Epidemiol, 157, 800-814.

6. ALTOMARE C, W. A. NORVELL, T. BJÖRKMAN, G. E. HARMAN, (1999),

Solubilization of phosphates and micronutrients by the plant growth promoting and

biocontrol fungus Trichoderma harzianum Rifai 1295-22, Appl. Environ.

Microbiol, 65, 2926–2933.

7. ANTOUN H, PRE´VOST D, (2005), Ecology of plant growth promoting

rhizobacteria, PGPR: Biocontrol and Biofertilization. Springer, Dordrecht, pp. 1–38.

8. ANTOUN H, PRÉVOST D, (2006), Ecology of plant growth promoting

rhizobacteria. In: PGPR: Biocontrol and Biofertilization, Siddiqui, Z.A. (ed.),

Springer, pp.1–38.

9. AS GH AR HN , ZA HIR ZA, AR S HAD M , KH A LI Q A,

( 20 02 ) , Rel a t i on s hi p between in vitro production of auxins by rhizobacteria an

d their growth-promoting activities in Brassica junceal, Biol Fertil Soils, 35

(23),1–237.

10. BABANA A H, ANTOUN H, (2006), Effect of Tilemsi phosphate rock-solubilizing

microorganisms on phosphorus uptake and yield of field-grown wheat (Triticum

aestivum L.) in Mali. Plant Soil 287, 51–58.

11. BAJPAI P.D, SUNDRA RAO, (1971), Phosphate Solubilizing Bacteria. Part 2,

Extracellular Production of Organic Acids by Selected Bacteria Solubilizing

Insoluble Phosphate, Soil Science and Plant Nutrition, Vol. 17, No.2, (April 1971),

pp. 44-45, ISSN 1747-0765.

12. BAKONYI T, DERAKHSHIFAR I, GRABENSTEINER E, NOWOTNY N, (2003),

Development and evaluation of PCR assays for the detection of Paenibacillus

larvae in honey samples: comparison with isolation and biochemical

characterization, Appl Environ Microbiol 69, 1504–1510.

13. BALAJI D.S, BASAVARAJA S, DESHPANDE R, MAHESH D.B, PRABHAKAR

B.K, VENKATARAMAN A, (2009), Extracellular biosynthesis of functionalized

silver nanoparticles by strains of Cladosporium cladosporioides fungus, Colloids

and surfaces B: biointerfaces, 68, 88- 92.

14. BANIK S, DEY BK, (1982), Available phosphate content of an alluvialsoil is

influenced by inoculation of some isolated phosphate-solubilizing microorganisms,

Plant Soil 69, 353 – 364.

15. BARKER K, (2003), Prespectives on plant and soil nematology, Ann. Rev.

Phytopathol, 41, 1-25.

16. BASHAN Y, HERNANDEZ JP, LEYVA LA, BACILIO M, (2002), Alginate

microbeads as inoculant carriers for plant growth-promoting bacteria, Biol Fert

Soils 35, 359–368.

17. BASHAN Y, HOLGUIN G, (1998), Proposal for the division of plant growth-

promoting rhizobacteria into two classifications: biocontrol-PGPB (plant growth-

promoting bacteria) and PGPB, Soil Biol Biochem 30, 1225–1228.

18. BEECH I.B, PAIVA M, CAUS M, COUTINHO C, (2001), Enzymatic Activity and

within Biofilms of Sulphate-Reducing Bacteria. In: Biofilm Community

Interactions: change or necessity?, 231-239, Boiline, ISBN 9780521812405 ,

Cardiff, U.K.

19. BEEVER R. E, BURNS, (1980), Phosphorus uptake, storage and utilization by

fungi, Advances in Botanical Research, 8,127-219.

20. BISWAS JC, LADHA JK, DAZZO FB, (2000), Rhizobial inoculation influences

seedling vigor and yield of rice. Agron J, 92, 880–886.

21. BORNEMAN J, P. W. SKROCH, K. M. O’SULLIVAN, J. A. PALUS, N. G.

RUMJANEK, J. L. JANSEN, J. NIENHUIS, E. W. TRIPLETT, (1996), Molecular

microbial diversity of an agricultural soil in Wisconsin, Appl. Environ.

Microbiol.62, 1935-1943.

22. BURD GI, DIXON DG, GLICK BR, (2000), Plant growth promoting bacteria that

decrease heavy metal toxicity in plants, Can J Microbiol 46, 237–245.

23. ÇAKMAKÇI R, ERAT M, ERDOĞAN, Ü. DÖNMEZ F, (2007), The influence of

plant growth-promoting rhizobacteria on growth and enzyme activities in wheat

and spinach plants, J. Plant Nutr. Soil Sci, 170, 288-295.

24. CAVALCANTI A, SHIRINZADEH B, FREITAS RA JR, HOGG T, (2008),

Nanorobot architecture for medical target identification, Nanotechnology 19 (1),

015103-15pp

25. CHABOT R, ANROUN H, CESCES MC, (1996), Growth promotion of maize and

lettuce by phosphate solubilizing Rhizobium leguminosarum biovar phaseoli, Plant

Soil 184, 31–121.

26. CHABOT R, ANTOUN H, CESCAS MP, (1996), Growth promotion of maize and

lettuce by phosphatesolubilizing Rhizobium leguminosarum biovar phaseoli, Plant

Soil 184, 311–321.

27. CHAI BO, WU YAN, LIU PENGMING, LIU BIAO, GAO MEIYING, (2011),

Isolation and phosphate-solubilizing ability of a fungus, Penicillium sp. from soil

of an alum mine, Journal of Basic Microbiology, Volume 51, issue 1, p. 5-14.

28. CHANG CH, YANG SS, (2009), Thermo-tolerant phosphate-solubilizing microbes

for multi-functional biofertilizer preparation, Bioresour Technol, 100, 1648–1658.

29. DAZZO FB, YANNI YG, RIZK R, DE BRUIJN FJ, RADEMAKER J, SQUARTINI

A, CORICH V, MATEOS P, MARTINEZ-MOLINA E (2000), Progress

inmultinational collaborative studies on the beneficial association between

Rhizobium Ieguminosarum by trifolii and rice, In: Ladha JK, Reddy PM (eds) The

quest for nitrogen fixation in rice. IRR1, Los Banos, Philippines, pp 167 – 189.

30. DEMEKE T, ADAMS R.P, (1994), The use of PCR-RAPD analysis of plant

taxonomy and evolution. in: Grifln, H.G., Grifln A.M. (Eds.), PCR Technology,

Current Innovations, CRC Press, Boca Raton, Florida, pp. 179–191.

31. DEUBEL A, GRANSEE, A, MERBACH W, (2000), Transformation of Organic

Rhizodeposits by Rhizoplane Bacteria and its Influence on the Availability of

Tertiary Calcium Phospate, Journal of Plant Nutrition and Soil Science, Vol. 163,

No.4, (August 2000), pp. 387-392, ISSN 1436-8730.

32. DEUBEL, A, W. MERBACH, (2005), Influence of Microorganisms on Phosphorus

Bioavailability in Soils, Microorganisms in Soils: Roles in Genesis and Functions.

Springer-Verlag, Berlin Heidelberg, Germany, p. 62.

33. DEY R, PAL KK, BHATT DM, CHAUHAN SM, (2004), Growth promotion and

yield enhancement of peanut (Arachis hypogaea L.) by application of plant growth-

promoting rhizobacteria, Microbiol. Res. 159, 371-394.

34. DIMKPA C, ALEŠ S, DIRK M, GEORG B, ERIKA K, (2008),

Hydroxamatesiderophores produced by Streptomyces acidiscabies E1 3 b i nd

n ic ke l a nd p rom ote g ro wt h i n c ow pea ( Vigna unguiculata L.) under

nickel stress, Can J Microb, 54, 163– 172.

35. DOBBELAERE S, VANDERLEYDEN J, OKON, Y, (2003), Plant growth-

promoting effects of diazotrophs in the rhizosphere, CRC Crit Rev Plant Sci 22,

107–149.

36. DODOR D. E, A. M. TABATABAI, (2003), Effect of cropping systems on

phosphatases in soils. J. Plant Nutr, Soil Sci. 166, 7–13.

37. DODOR D.E, TABATABAI M.A, (2003), Amidohydrolases in soils as affected by

cropping systems, Appl. Soil Ecol.24, 73–90.

38. DORAU B, ARANGO R, GREEN F, (2004), Proceedings of the 2nd Wood-Frame

Housing Durabili and Disaster Issues Conference, Forest Products Society, Las

Vegas,.

39. DURÁN N, PRISCYLA D, OSWALDO M, ALVES L, (2005), Mechanistic aspects

of biosynthesis of silver nanoparticles by several Fusarium oxysporum strains,

Journal of Nanobiotechnology, 1186/1477-3155-3-8

40. Duran N, Marcato D.P, Alves L.O, De Souza G, Esposito E, (2005), Mechanical

aspect of biosynthesis of silver nanoparticles by several Fusarium oxysporum

strains, J, Nanobiotechnology, 3, 8-15.

41. DUTTON M.V, EVANS C.S, (1996), Oxalate production by fungi: Its role in

pathogenicity and ecology in the soil environment, Can. J. Microbiol, 42,881–895.

42. EDEL V, STEINBERG C, AVELANGE I, LAGUERRE G, ALABOUVETTE C,

(1995), Comparison of three molecular methods for the characterization of

Fusarium oxysporum strains, Phytopathology 85, 579–85.

43. EDI PREMONO M, MOAWAD A. M, AND VLEK P. L. G, (1996), Effect of

phosphate-solubilizing Pseudomonas putida on the growth of maize and its survival

in the rhizosphere, Indones. J. Crop Sci, 11, 13Ð23.

44. E GAM BE R D IY E V A D , (2 0 05) , Pla nt g r owt h

p r om ot i ng r hi zo b z t e r i a i so l a t e d f r om a ca l s i s o l i n sem i a r id

r eg io n o f U zbe k i s t a n: b i oc hem ica l c ha ra c te r i za t io n

a n d effectiveness, J Plant Nutr. SoilSci, 168, 94 – 99.

45. ELLSWORTH D. L, RITTENHOUSE K. D. AND HONEYCUTT R. L, (1993),

Artifactual variation in randomly amplied polymorphic DNA banding patterns, Bio

Techniques, 14, 214–216.

46. EZAWA T, SMITH S.E, SMITH F.A, (2002), P metabolism and transport in AM

fungi, Plant Soil 244, 221–230.

47. FANKEM H, D. NWAGA, A. DEUBEL, L. DIENG, W. MERBACH AND F. X.

ETOA, (2006), Occurrence and functioning of phosphate solubilizing

microorganisms from oil palm tree (Elaeis guineensis) rhizosphere in Cameroon,

African J. Biotech. 5, 2450-2460.

48. FENG K, LU HM, SHENG HJ, WANG XL, (2004), Effect of organic ligands on

biological availability of inorganic phosphorus in soils, Pedosphere, mao j 14, 85–

92.

49. FENG X, L.T. OU, A. OGRAM, (1997), Plasmid mediated mineralization of the

insecticide carbofuran by Sphingomonas sp. CFO6. Appl. Environ, Microbiol. 63,

1332-1337.

50. FENG QL, WU J, CHEN GQ, CUI FZ, KIM TN, KIM JO, (2000), A mechanistic

study of the antibacterial effect of silver ions on Escherichia coli and

Staphylococcus aureus, J Biomed Mater Res, 52, 662–668.

51. FOX, R, N. B. COMERFORD, W. W. MCFEE, (1990), Phosphorus and aluminium

release from a spodic horizon mediated by organic acids, Soil Sci. Soc. Am. J, 54,

1763-1767.

52. FROSTEGARD A, S. COURTOIS, V. RAMISSE, S. CLERC, D. BERNILLON, F.

LE GALL, P.JEANNIN, X. NESME, P. SIMONT, (1999), Quantification of bias

related to the extraction of DNA directly from soils. Appl. Environ. Microbiol. 65,

5409-5420.

53. GAMALERO E, LINGUA G, CAPRI FG, FUSCONI A, BERTA G,LEMANCEAU

P, (2004), Colonization pattern of primary tomato roots by Pseudomonas

fluorescens A6RI characterized by dilution plating, flow cytometry, fluorescence,

confocal and scanning electron microscopy, FEMS Microbiology Ecology 48, 79-

87.

54. GAUR C, GAIND S, (1999), Phosphate solubilizing microorganisms-An overview.

In Agromicrobes, Current trends in life sciences, Today and tomorrows publishers,

New Delhi.

55. GAUR, A.C AND GAIND, S, (1983), Microbial solubilization of phosphates with

particular reference to iron and aluminum phosphate , Sci.Cult. 49, 110.

56. GAVRIELI Y, SHERMAN Y, BEN-SASSON SA, (1992), Identification of

programmed cell death in situ via specific labeling of nuclear DNA fragmentation,

J cell Biol 119, 493–501.

57. GEETIKA AND NANDAN, (2008), ‘A study of socio-economic influences on

perceptions of gender equality in India’, Integral Review, Integral University,

Lucknow, pp 13-15.

58. GERHARDSON B, (2002), Biological substitutes for pesticides, Trends Biotechnol,

20, 338-343.

59. GERRETSEN, F. C, (1948), The influence of microorganisms on the phosphate

intake by the plant, Plant Soil, 1, 51-81.

60. GLICK BR, KARATUROVIC DM, NEWELL PC, (1995), A novel procedure for

rapid isolation of plant growth promoting Pseudomonas, Can. J Microbiol, 41,533–

536.

61. GLICK BR, PATTEN CL, HOLGIN G, PENROSE DM, (1999), Biochemical and

genetic mechanisms used by plant growth promoting bacteria, Imperial College

Press, London, 267 p.

62. GOLDSTEIN A H , LIU S T, (1987), Molecular cloning and regulation of a

mineral phosphate solubilizing gene from Erwinia herbicola, Biotechnology 5, 72–

74.

63. GOLDSTEIN A. H, (1994), Involvement of the quinoprotein glucose

dehydrogenase in the solubilization of exogenous mineral phosphates by gram-

negative bacteria, Phosphate in Microorganisms: Cellular and Molecular Biology,

Washington, DC, 197–203.

64. GOLDSTEIN A.H, (2000), Bioprocessing of Rock Phosphate Ore: Essential

Technical Considerations for the Development of a Successful Commercial

Technology, Proceedings of 4th International Association for Technical Conference,

pp. 220-242, ISBN 1-57498-079-3, New Orieans, USA, October 1-4, 2000.

65. GOLDSTEIN AH, ROGERS RD, MEAD G, (1993), Mining by microbe,

Biotechnology 11, 1250–1254.

66. GROISMAN EA, CASTILLO BA, CASADABAN MJ, (1984) , In vivo DNA

cloning and adjacent gene fusing with a mini-Mulac bacteriophage containing a

plasmid replicon, Proc Natl Acad Sci USA, 81, 1480–3.

67. GYANESHWAR P, KUMAR, N.J. PAREKA, L.J, PODLE, P.S, (2002), Role of Soil

Microorganisms in Improving P Nutrition of Plants. Plant and Soil, Vol. 245, No. 1,

(August 2002), pp. 83-93, ISSN 1573-5036.

68. GYANESHWAR P, NARESH KUMAR G, PAREKH LJ, POOLE PS, (2002), Role

of soil microorganisms in improving P nutrition of plants, Plant Soil , 245, 83–93.

69. GYANESHWAR P, PAREKA L.J, ARCHANA G, PODLE P.S, COLLINS M.D,

HUSTON R.A, NARESH K.G, (1999), Involvement of a Phosphate Stravation

Inducible Glucose Dehydrogenase in Soil Phosphate Solubilization by

Enterobacter asburiae, FEMS Microbiology Letters, Vol. 171, No. 2, (February

1999), pp. 223-229, ISSN 0378-1097.

70. HARTMAN KB, WILSON LJ, ROSENBLUM MG, (2008). Detecting treating

cancer with nanotechnology, Mol Diagn Ther, 12, 1–14.

71. HE, Z. L, J. ZHU, (1988) , Microbial utilization and transformation of phosphate

adsorbed by variable charged minerals, Soil Biol. Biochem, 30, 917-923.

72. HENRI F, LAURETTE N. N, ANNETTE D, JOHN Q, WOLFGANG M,

FRANÇOIS-XAVIER E, N. DIEUDONNÉ, (2008), Solubilization of inorganic

phosphates and plant growth promotion by strains of Pseudomonas fluorescens

isolated from acidic soils of Cameroon, African J. Microbiol. Res. 2, 171-178.

73. HENRY R, (1997), Molecular markers in plant improvement In: Practical

applications of Plant , Molecular Biology, Chapman and Hall, London., 99–132.

74. HILDA R, FRAGA R, (2000), Phosphate solubilizing bacteria and their role in

plant growth promotion, Biotechnol. Adv, 17, 319-359.

75. HINSINGER P, (2001), Bioavailability of Soil Inorganic P in the Rhizosphere as

Affected by Root-induces Chemical Changes: A Review, Plant and Soil, Vol. 237,

No.2, (December 2001), pp. 173-195, ISSN 0032-0079X.

76. HUSSAIN, S. M, HESS, K. L, GEARHART, J. M, GEISS K. T, SCHLAGER, J. J,

(2005), In Vitro Toxicity of Nanoparticles in BRL 3A Rat Liver Cells, Toxicol, In

Vitro, 19, 975–983.

77. HUSTON M. A, (1994), Biological Diversity: The Coexistence of Species on

Changing Landscapes, Cambridge University Press, Cambridge.

78. ILLMER P, BARBATO A, SCHINNER F, (1995), Solubilization of hardly-soluble

AlPO4 with P-solubilizing microorganism, Soil Biol. Biochem, 27, 265–270.

79. ILLMER P, F. SCHINNER, (1995), Solubilization of inorganic phosphates by

microorganisms isolated from forest soil, Soil Biol. Biochem, 24, 389-395.

80. JEFFRIES P, GIANINAZZI S, PEROTTO S, TURNAU K, (2003), The contribution

of arbuscular mycorrhizal fungi in sustainable maintenance of plant health and

soil fertility, Biol Fert Soils 37, 1–16.

81. JIANG CY, SHENG XF, QIAN M, WANG QY , (2008), Isolation and

characterization of a heavy metal resistant Burkholderia sp. from heavy metal-

contaminated paddy field soil and its potential in promoting plant growth and

heavy metal accumulation in metal-polluted soil, Chemosphere, 72, 157–164.

82. JONES D, (1998), Organic acids in the rhizosphere - a critical review, Plant Soil

205, 25-44.

83. JOSHI S, RANJEKAR, P, GUPTA, V, (1999), Molecular markers in plant genome

analysis, Curr Sci 77, 230–240.

84. JUCK D, CHARLES T, WHYTE L.G, GREER C.W, (2000), Polyphasic microbial

community analysis of petroleum hydrocarbon-contaminated soils from two

northern Canadian communities, FEMS Microbiol. Ecol., 33, 241–249.

85. KATZNELSON H, BOSE .B, (1959), Metabolic activity and phosphate dissolving

capability of bacterial isolates from wheat roots, rhizosphere and non-rhizosphere

soil, Can. J Microbiol, 5- 79-85.

86. KHALID A, ARSHAD M, ZAHIR ZA, (2001), Factor affecting auxin biosynthesis

by wheat a rice rhizobacteria, Pak J Soil Sci 21, 11 – 18.

87. KHAN M. S, A. ZAIDI, P. A. WANI, (2007), Role of phosphate-solubilizing

microorganisms in sustainable agriculture - A review, Agron. Sustain. Dev. 27, 29-

43.

88. KHIARI L, PARENT LE, (2005), Phosphorus transformations in acid light

textured soils treated with dry swine manure, Can. J. Soil Sci. Soc, 85, 75-87.

89. KIER LD, WEPPELMAN R, AMES BN, (1977), Resolution and purification of 3

phosphatases of Salmonella typhimurium, J Bacteriol, 130, 411–9.

90. KIM K.V, JORDAN D, MCDONALD G.A, (1998), Effect of Phosphate

Solubilizing Bacteria and Vesicular-Arbuscular Mycorrhizae on Tamoto Growth

and Soil Microbial Activity, Biology and Fertility of Soils, Vol.26, No. 2, (December

1997), pp. 79-87, ISSN 1432-0789.

91. KIM, K.Y, JORDAN, D, MCDONALD, D, (1997), Solubilization of Hydroxyapatite

by Enterobacter Agglomerans and Cloned Escherichia in Culture Medium, Biology

and Fertility of Soils, Vol. 24, No. 4, (May 1997), pp. 347-352, ISSN 1432-0789.

92. KPOMBLEKOU K, TABATABAI MA, (1994), Effect of organic acids on release

of phosphorus from phosphate rocks, Soil Sci 158, 442–453.

93. LAM C-W, JAMES JT, MCCLUSKEY R, (2004), Pulmonary toxicity of single-wall

carbon nanotubes in mice 7 and 90 days after intratracheal instillation, Tox Sc, 77,

126–34.

94. LAVAN DA, MCGUIRE T, LANGER R, (2003). Small-scale systems for in vivo

drug delivery, Nat Biotechnol. 21 (10), 1184–1191

95. LEACH AW, MUMFORD JD, (2011), Pesticide Environmental Accounting: A

decision-making tool estimating external costs of pesticides, Journal of Consumer

Protection and Food Safety, Vol, 6, Pages, 21-26.LI, S.; SHEN, Y.; XIE,A.; YU, X.;

QUI, L.; ZHANG, L. AND ZHANG, Q, (2007), Green synthesis of silver

nanoparticles using Capsicum annum L. extract, Green Chemistry, 9, 852-858.

96. LEBEL CP, ISCHIROPOULOS H, BONDY SC, (1992), Evaluation of the probe

2’,7’- dichlorofluorecein as an indicator of reactive oxygen species formation and

oxidative stress, Chem Res Toxicol, 5, 227-231.

97. LIAO R.S, RENNIE R.P, TALBOT J.A, (1999), Assessment of the effect of

amphotericin B on the vitality of Candida albicans. Antimicrobial agents and

chemotherapy, Vol. 43, No. 5, 1034-1041.

98. LIAU SY, READ DC, PUGH WJ, FURR JR, RUSSELL AD, (1997), Interaction of

silver nitrate with readily identifiable groups: relationship to the antibacterial

action of silver ions, Lett Appl Microbiol, 25(4), 279–283.

99. LOWRY O.H, N.J. ROSEBROUGH, A.L. FARR, R.J. RANDALL, (1951), Protein

Measurement with the Folin Phenol Reagent., J. Biol. Chem. 193, 265-275.

100. M. GERICK, A. PINCHES, (2006), Biological synthesis of metalnanoparticles,

Hydrometallurgy, vol. 83, no. 1–4, pp. 132–140.

101. MALBOOBI MA, BEHBAHANI M, MADANI H, OWLIA P, DELJOU A,

YAKHCHALI B, MORADI M, HASSANABADI H, (2009), Performance

evaluation of potent phosphate solubilizing bacteria in potato rhizosphere, World J

Microbiol Biotechnol , 25, 1479–1484.

102. MATIAS SR, PAGANO MC, CARVALHO-MUZZI F, OLIVEIRA CA, ALMEIDA-

CARNEIRO A, HORTA SN, SCOTTI MR , (2009), Effect of rhizobia, mycorrhizal

fungi and phosphate-solubilizing microorganisms in the rhizosphere of native

plants used to recover an iron ore area in Brazil, Eur J Soil Biol ,45, 259–266.

103. MIKANOVA O, NOVAKOV J, (2002), Evaluation of the P-solubilizing activity of

soil microorganisms and its sensitivity to soluble phosphate, Rostlinna Vyroba 48

(9) 397-400.

104. MILLER D. N, J. E. BRYANT, E. L. MADSEN, W. C. GHIORSE, (1999),

Evaluation and optimization of DNA extraction and purification procedures for

soil and sediment samples, Appl. Environ. Microbiol. 65, 4715-4724.

105. MOGHIMI, A, M. E. TATE, (1978), Does 2-ketogluconate chelate calcium in the

pH range 2.4 to 6.4, Soil Biol. Biochem, 10, 289-292.

106. MOHANPURIA P, RANA N.K, YADAV S.K, (2008), Biosynthesis of

nanoparticles: Technological concepts and future applications, J. Nanopart. Res,

10, 507–517.

107. MORE M. I, HERRICK J. B, SILVA M. C, GHIORSE W. C, E. L. MADISEN,

(1994), Quantitative cell lysis of indigenous microorganisms and rapid extraction

of microbial DNA from sediment, Appl. Environ. Microbiol, 60, 1572-1580.

108. MOSMANN F, (1983), Rapid calorimetric assay for cellular growth and survival:

application to proliferation and cytotoxicity assay, J Immunol. Methods, 65, 55–63.

109. MOYER C. I, F. C. DOBBS, D. M. KARL, (1994), Estimation of diversity and

community structure through restriction fragment length polymorphism

distribution analysis of bacterial 16S rRNA genes from a microbial mat at an active

hydrothermal vent system, Appl. Environ. Microbiol, 60, 871-8.

110. M U R AT O V A A Y U , T U R KO V SK AY A O V , AN T O N Y U K LP , M A

K AR O V OE , POZDNYAKOVA LI, IGNATOV VV, (2005), Oil-oxidizing

potential of associative rhizobacteria of the genus Azospirillum, Microbiology, 74,

210 – 215.

111. MUYZER G, (1999), DGGE/TGGE a method for identifying genes from natural

ecosystems, Curr. Opin. Microbiol, 2, 317 – 322.

112. MUYZER G, SMALLA K, (1999), Application of denaturing gradient gel

electrophoresis (DGGE) and temperature gradient gel electrophoresis in microbial

ecology, Antonie van Leeuwenhock , 73,127 – 141.

113. NAHAS E, BANZATTO D.A, ASSIS L.C, (1990), Fluorapatite solubilization by

Aspergillus niger in vinasse medium. Soil Biol. Biochem, 22, 1097–1101.NAHAS,

E, (1996), Factors Determining Rock Phosphate Solubilization by Microorganisms,

World Journal of Microbiology and Biotechnology, Vol. 12, No. 6, (November

1996), pp. 567-572.

114. NAHAS, E., (1996), Factors determining rock phosphate solubilization by

microorganism isolated from soil, World J. Microb. Biotechnol. 12, 18-23.

115. NEI M, LI W-H, (1979), Mathematical model for studying genetic variation in

terms of restriction endonucleases, Proceedings of the National Academy of Science

USA 76, 5269-5273.

116. OMAR S. A, (1998), The role of rock-phosphate-solubilizing fungi and vesicular–

arbuscular mycorrhiza (VAM) in growth of wheat plants fertilized with rock

phosphate, World J. Microbiol. Biotechnol, 14, 211-218.

117. ORTUNO A, HERNANSAEZ A, NOGUERA J, MORALES V, ARMERO T,

(1978), Phosphorus solubilizing effect of Aspergillus niger and Pseudomonas

fluorescens. Microbiologı´a Espan˜ola , 31, 113–120.

118. OTANI T, N. AE, H. TANAKA, (1996), Phosphorus (P) uptake mechanisms of

crops grown in soils with low P status. II. Significance of organic acids in root

exudates of pigeonpea, Soil Sci Plant Nutr, 42, 553-560.

119. PAL S, TAK Y.K, SONG J.M., (2007), Does the antibacterial activity of silver

nanoparticles depend on the shape of the Nanoparticle? A study of the gram-

negative bacterium Escherichia coli. , Appl. Environ. Microbiol, 73, 1712-1720.

120. PALLERONI N. J, M. DOUDOROFF, R. Y. STANIER, R. E. SOLANES, M.

MANDEL, (1970), Taxonomy of the aerobic pseudomonads: The properties of the

Pseudomonas stutzeri group, J. Gen. Microbiol, 60, 215–231.

121. PARASHAR, V, PRASHAR, R, SHARMA, B, PANDEY, A.C, (2009), Parthenium

leaf extract mediated synthesis of silver nanoparticles: a novel approach towards

weed utilization, Digest journal of nanomaterials and biostructures, 4(1), 45-50.

122. PATRA CR, BHATTACHARYA R, WANG E, KATARYA A, LAU JS, DUTTA S,

MUDERS M, WANG S, BUHROW SA, SAFGREN SL, YASZEMSKI MJ, REID

JM, AMES MM, MUKHERJEE P, MUKHOPADHYAY D, (2008) , Targeted

delivery of gemcitabine to pancreatic adenocarcinoma using cetuximab as a

targeting agent, Cancer Res, 68, 1970–8.

123. PENNER J.E, H. EDDLEMAN, T. NOVAKOV, (1993), Towards the development

of a global inventory for black carbon emissions, Atmos. Environ, 27, 1277-1295.

124. PIKOVSKAYA R. I, (1948), Mobilization of Phosphate in Soil in Connection With

Their Vital Activities of Some Microbial Species, Microbiologiya, 17, 362-370.

125. POND JL, EDDY CK, MACKENZIE KF, CONWAY T, BORECKY DJ, INGRAM

LO, (1989) , Cloning, sequencing and characterization of the principal acid

phosphatase, phoC product, from Zymomonas mobilis, J Bacteriol, 171, 767–74.

126. POONGUZHALI S, MUNUSAMY M, SA T, (2008), Isolation and identification of

phosphate solubilizing bacteria from Chinese cabbage and their effect on growth

and phosphorus utilization of plants, J Microbiol Biotechnol, 18, 773–777.

127. PRADEL E, BOQUET PL, (1988), Acid phosphatases of Escherichia coli:

molecular cloning and analysis of agp, the structural gene for a periplasmic acid

glucose phosphatase, J Bacteriol, 170, 4916–23.

128. RAI M, YADAV A, GADE A. (2009), Silver nanoparticles as a new generation of

antimicrobials, Biotech. Advances, 27, 76-83.

129. RAJANKAR, P.N., TAMBEKAR, D.H., WATE, S.R., (2007), Study of phosphate

solubilization efficiencies of fungi and bacteria isolated from saline belt of Purna

river basin. Agriculture and Sciences, 3(6), 701-703.

130. RAMAMOORTHY V, VISWANATHAN R, RAGUCHANDER T, PRAKASANV,

SAMIYAPPAN R, (2001), Induction of systemic resistance by plant growth

promoting rhizobacteria in crop plants against pests and diseases, Crop Protection,

20, 1-11.

131. RAMSING N.B, FOSSING H, FERDELMAN T.G, ANDERSEN F, THAMDRUP

B. , (1996), Distribution of bacterial populations in a stratified fjord (Mariager

Fjord, Denmark) quantified by in situ hybridization to chemical gradient in the

water column, Appl. Environ. Microbiol, 62, 1392–1404.

132. REYES I, A. VALERY, Z. VALDUZ, (2007), Phosphate solubilizing

microorganisms isolated from rhizospheric and bulk soils of colonizer plants at an

abandoned rock phosphate mine, In: E. Velázquez and C. Rodríguez-Barrueco

(eds.), First International Meeting on Microbial Phosphate Solubilization, pp. 69-

75.

133. RICCIO ML, ROSSOLINI GM, LOMBARDI G, CHIESURIN A, SATTA G, (1997),

Expression cloning of different bacterial phosphatase-encoding genes by

histochemical screening of genomic libraries onto an indicator medium containing

phenolphthalein diphosphate and metyl green, J Appl Bacteriol, 82, 177–85.

134. RICHARDSON A, (2001), Prospects for using soil microorganisms to improve the

acquisition of phosphorus by plants, Aust J Plant Physiol, 28, 897–907.

135. ROD M, (2008), Sizing up targets with nanoparticles, Nature nanotechnology 3 (1),

12–13.

136. RODRIGUEZ H, FRAGA R, (1999), Phosphate solubilizing bacteria and their role

in plant growth promotion, Biotechnol Adv, 17, 319–339.

137. RODRIGUEZ H, FRAGA R, GONZALEZ T, BASHAN Y, (2006), Genetics of

phosphate solubilization and its potential applications for improving plant growth-

promoting bacteria, Plant Soil 287, 15–21.

138. RODRIGUEZ H, GONZALEZ T, GOIRE I, BASHAN Y, (2004), Gluconic acid

production and phosphate solubilization by the plant growth-promoting bacterium

Azospirillum spp., Naturwissenschaften, 91, 552–555.

139. ROJAS A, HOLGUIN G, GLICK BR, BASHAN Y , (2001), Synergism between

Phyllobacterium sp. (N2-fixer) and Bacillus licheniformis (P-solubilizer), both from

a semiarid mangrove rhizosphere, FEMS Microbiol Ecol , 35, 181–187.

140. ROOSE-AMSALEG C. L. E. GARNIER-SILLAM, M. HARRY, (2001), Extraction

and purification of microbial DNA from soil and sediment samples, Appl. Soil Ecol.

18, 47-60.

141. ROSE R.E, (1957), Techniques of determining the effect of micro-organisms on

insoluble inorganic phosphates, N. Z. J. Sci. Technol., 38-780.

142. RUDRESH D.L., M.K. SHIVAPRAKASH, R.D. PRASAD, (2004) , Effect of

combined application of Rhizobium, phosphate solubilizing bacterium and

Trichoderma spp. on growth, nutrient uptake and yield of chickpea (Cicer

aritenium L.), Applied Soil Ecology 28, 139-146.

143. SAGOE CI, ANDO T, KOUNO K, NAGAOKA T, (1998), Effects of organic acid

treatment of phosphate rocks on the phosphorus availability to Italian ryegrass.

Soil Sci. Plant Nut. 43, 1067-1072.

144. SAHIN F, C¸ AKMAKC¸I R, KANTAR F, (2004), Sugar beet and barley yields in

relation to inoculation with N2-fixing and phosphate solubilizing bacteria, Plant

Soil 265, 123–129.

145. SAMBROOK J, FRITSCH EF, MANIATIS T, (1989), Molecular Cloning: A

Laboratory Manual (2nd Edition), Cold Spring Harbor Laboratory, NY.

146. SATTAR M.A, GAUR A.C, (1985), Characterization of phosphate dis-solving

microorganisms isolated from some Bangladesh soil samples, Bangladesh Journal

of Microbiology 2, 22-28.

147. SHARMA V, KUMAR V, ARCHANA G, KUMAR GN, (2005) , Substrate

specificity of glucose dehydrogenase (GDH) of Enterobacter asburiae PSI3 and

rock phosphate solubilization with GDH substrates as C sources, Can J Microbiol,

51, 477–482.

148. SHENG X.F, HUANG W.Y, (2002), Mechanism of potassium release from feldspar

affected by the strain NBT of silicate bacterium, Acta Pedol. Sin, 39, 863–871.

149. SIDDIQUI, ZA, MAHMOOD I, (1999), Role of bacteria in the management of

plant parasitic nematodes: a review, Bioresource Technol 69, 167–179.

150. SRIDEVI N, SRILAKSHMI. C, SINGH S, (2013), Comparative studies on growth

parameters and physio-chemical analysis of Pleurotus ostreatus and Pleurotus

florida, Asian Journal of Plant Science and Research, 3, 163-169

151. STAUNTON S, LEPRINCE F, (1996), Effect of pH and some organic anions on

the solubility of soil phosphate: implications for P bioavailability, Eur. J. Soil Sci.

47, 231-239.

152. STEPHENJ, M. S. JISHA, (2009), Buffering reduces phosphate solubilizing ability

of selected strains of bacteria, World J. Agric. Sci. 5, 135-137.

153. STEVENSON F.J, (2005), Cycles of Soil: Carbon, Nitrogen, Phosphorus, Sulfur,

Micronutrients, 471-32071-4.

154. SUMAN A, SHASANY A.K, SINGH M, SHAHI H.N, GAUR, A, KHANUJA,

S.P.S, ( 2001 ), Molecular assessment of diversity among endophytic diazotrophs

isolated from subtropical Indian sugarcane, World Journal of Microbiology and

Biotechnology 17, 39–45.

155. SUNDARA B, V. NATARAJAN, K. HARI, (2002), Influence of phosphorus

solubilizing bacteria on the changes in soil available phosphorus and sugarcane

yields, Field Crops Res. 77, 43-49.

156. TANG C, Z. RENGEL, (2003), Role of plant cation/anion uptake ratio in soil

acidification. In: Z. Rengel (Ed.), Handbook of Soil Acidity, pp. 57-81.

157. TAO GC, TIAN SJ, CAI M Y, XIE GH, (2008), Phosphate-solubilizing and -

mineralizing abilities of bacteria isolated from soils, Pedosphere, 18, 515–523.

158. TESKE, A, WAWER C, MUYZER, G, RAMSING N.B, (1996), Distribution of

sulfate-reducing bacteria in a stratified fjord (Mariager Fjord, Denmark) as

evaluated by most-probable number counts and denaturing gradient gel

electrophoresis of PCR-amplified ribosomal DNA fragments, Appl. Environ.

Microbiol, 62, 1405-1415.

159. TORSVIK V, GOKSOYER J, F. L. DAAE (1990), High diversity in DNA of soil

bacteria, Appl. Environ. Microbiol, 56, 782-787.

160. TREVORS J. T, LEE H, COOK S, (1992), Direct extraction of DNA of soil bacteria,

Microbiol. Releases 1, 111-115.

161. TSAI Y. L, OLSON B. H, (1992), Rapid method for separation of bacterial DNA

from humic substances in sediments for polymerase chain reaction, Appl. Environ.

Microbiol, 58, 2292-2295.

162. UNIVERSITY OF WATERLOO, (2010), Nanotechnology in Targeted Cancer

Therapy, http://www.youtube.com/watch?v=RBjWwlnq3cA.

163. VASSILEVA M, VASSILEV N, FENICE M, FEDERICI F, (2001), Immobilized cell

technology applied in solubilization of insoluble inorganic (rock) phosphate and P

plant acquisition, Bioresource Technol. 79 , 263-271.

164. VILLEGAS J, FORTIN J. A, (2002), Phosphorus solubilization and pH changes as

a result of the interactions between soil bacteria and arbuscular mycorrhizal fungi

on a medium containing NO3 as nitrogen source, Can. J. Bot. 80, 571-576.

165. WANI P.A, KHAN M.S, ZAIDI A, (2007), Synergistic effect of the inoculation

with nitrogen-fixing and phosphate-solubilizing rhizobacteria on performance of

field-grown chickpea, Journal of Plant Nutrition and Soil Science, 170, 283-287.

166. WHITE TJ, BRUNS T, LEE S, TAYLOR J ,(1990), Amplification and direct

sequencing of fungal ribosomal RNA genes for phylogenetic, In: PCR Protocols: a

guide to methods and applications. (Innis MA, Gelfand DH, Sninsky JJ, White TJ,

eds). Academic Press, New York, USA: 315–322.

167. WHITELAW, M.A, (2000), Growth Promotion of Plants Inoculated with

Phosphate Solubilizing Fungi, Advances in Agronomy, Vol. 69, (December 1999),

pp. 99-151, ISSN 0065-2113.

168. WILLIAMS J.G.K, KUBELIK A.R, LIVAK KJ., RAFALSKI J.A, TINGEY S.V,

(1990), DNA polymorphisms amplified by arbitrary primers are useful as genetic

markers, Nucleic Acid Res 18, 6531-6535.

169. WRIGHT J.B, LAM K, HANSEN D, BURRELL R. E, (1999), Efficacy of topical

silver against fungal burn wound pathogens, Am. J. Infest. Control 27, 344-350.

170. XIA X, BOLLINGER J, OGRAM A, (1995), Molecular genetic analysis of the

response of three soil microbial communities to the application of 2, 4-D, Mol.

Ecol. 4, 17–28.

171. XIE, H, PASTERNAK J. J, GLICK B. R, (1996), Isolation and characterization of

mutants of the plant growth promoting rhizobacterium Pseudomonas putida GR12-

2 that overproduce indoleacetic acid, Curr. Microbiol, 32, 67-71.

172. YADAV B.K, TARAFDAR J.C, (2007), Ability of Emericella rugulosa to Mobilize

Unavailable P Compounds during Pearl Millet (Pennisetum glaucum L.) Crop

under Arid Condition, Indian Journal of Microbiology, Vol. 41, No. 1, (March 2007),

pp. 57-63.

173. YADAV R.S, TARAFDAR J.C, (2001), Influence of Organic and Inorganic

Phosphorous Supply on the Maximum Secretion of Acid Phosphatase by Plants,

Biology and Fertility of Soils, Vol. 34, No. 3, (September 2001), pp.140-143.

174. YEATES C, M. R. GILLINGS, (1998), Rapid purification of DNA from soil for

molecular biodiversity analysis, Lett. Appl. Microbiol, 27, 49-53.

175. YEATES, C, M. R. GILLINGS, A. D. DAVISON, N. ALTAVILLA, A. VEAL,

(1997), PCR amplification of crude microbial DNA extracted from soil, Lett.

Appl.Microbiol, 25, 303-307.

176. YLINIEMI K, VAHVASELKA, M, (2008), Antimicrobial activity of colloidal silver

nanoparticles prepared by sol-gel method, Chem. 18, 199.

177. YI Y, HUANG W, GE Y, (2008), Exo polysaccharide: a novel important factor in

the microbial dissolution of tricalcium phosphate, World J Microbiol Biotechnol 24,

1059–1065.

178. ZAHIR Z.A, ARSHAD M, FRANKENBERGER W.T, (2004), Plant growth

promoting rhizobacteria: applications and perspectives in agriculture, Adv. in

Agron. 81, 97-168.

179. ZHOU J, M. A. BRUNS, TIEDJE, (1996), DNA recovery from soils of diverse

composition, Appl. Environ. Microbiol, 62, 316-322.

List of Publications

1. Paper Published/ Book Chapters: (Total: 4)

1. Faria Fatima, Ira Chaudhary, J Ali, S Rastogi, N Pathak. Exploring microbial diversity

for development National conference on biodiversity, development and poverty

alleviation 22 may, 2010.

2. Faria Fatima, Ira Chaudhary, Jasarat Ali, Smita Rastogi, Neelam Pathak. Microbial

DNA extraction from soil by different methods and its PCR amplification. Biochem.Cell

Arch. 2011, 11: 1-6.

3. Ira Chaudhary, Faria Fatima, Rolee Sharma, Smita Rastogi and Neelam Pathak.

Isolation of a Noval Laccase- producing Fungus from litter in Upper Soil Horizon, Braz.

J. Microbial. 2011. (Communicate).

4. Ira Chaudhary, Faria Fatima, Smita Rastogi, Neelam Pathak, Zeeshan. Isolation and

Characterization of microbes with Phosphate Solubilizers and lignin degrading abilities.

Biochem. Cell. Arch. Vol. 11, No.2, 2011.

Abstract: (Total: 15)

1. Faria Fatima, Nilesh Kumar, Srika Singh ,Preeti Bajpai and Neelam Pathak.

Biological Synthesis of Nanoparticles by Novel Phosphate solubilizsing Fungus

(Bipolaris tetramera) and their antibacterial activity. Alumni Meet and National

Seminar on “Frontier Agriculture-2013”. Deva University Of Agriculture and

technology, Kumarganj Faizabad .April 20-21, 2013.

2. Faria Fatima, Jiya Naseem, Erum Sheikh, Pawan Kushwaha, Preeti Bajpai, Neelam

Pathak. Extracellular synthesis of silver and gold Nanoparticles bu Bipolaris

tetramera and their antibacterial activity. Innovations in sciences for better

tomorrow, “LUSCON 2013” 20-21 March 2013 Lucknow (Awarded II Prize in

poster presentation)

3. Faria Fatima, Erum Sheikh, Pawan kushwaha, Priyanka verma, Priti bajpai,

Neelam Pathak and A.K Srivastava. Fungus mediated extracellular synthesis of

silver and gold nanoparticles and their antibacterial activity. National seminar on

stress, development and adaptation: Biochemical Basis and Biotechnological

Approaches 15-16 March 2013 Lucknow.

4. Jasarat Ali, Faria Fatima and Neelam Pathak. Screening. Biochemical and

Molecular Characterization of bacteria having (1-Aminocyclopropane-1-

Carboxylate)-Deaminase Producing Potential. Alumni Meet and National Seminar

on “Frontier Agriculture-2013”. Deva University of Agriculture and technology,

Kumarganj Faizabad .April 20-21, 2013.

5. Faria Fatima. Biodiversity Analysis of Phosphate Solubilizers at biochemical and

Molecular level. Bright international conference and events on World congress

biotechnology, 4-6 May 2012.

6. Faria Fatima, Ira Chaudhary, Neelam Pathak Smita Rastogi and Rolee Sharma.

Isolation, Biochemical and Molecular Characterization of microbes with Phosphate

solubilizing abilities. International Conference on Microbial, Plant and Animal

research (ICMPR-2012), March 29-31, 2012 Jaipur.

7. Faria Fatima, Jasarat ali and Neelam Pathak. Isolation , biochemical and molecular

characterization of microbes with phosphate solubilizing abilities. Department of

Biotechnology, Integral University, Nov 11, 2012 Lucknow.

8. Jasarat Ali, Faria Fatima, Farina Mujeeb and Neelam Pathak. Isolation of ACC

Deaminase Producing Microrganisms and their Biochemical and Molecular

Characterization. Department of Biotechnology, Integral University, Nov 11, 2012

Lucknow.

9. Ira Chaudhary, Faria Fatima, Neelam Pathak Smita Rastogi and Rolee Sharma.

Isolation of Lignin Peroxidase –Producing lignin degrading fungus from soil.

International Conference on Microbial, Plant and Animal research (ICMPR-2012),

March 29-31, 2012 Jaipur.

10. Ira Chaudhary, Faria Fatima ,Neelam Pathak, Smita Rastogi, Rolee Sharma.

Isolation of Lignin Peroxidase- producing Plant Pathogenic Fungus from the effluent

sample: Planetary Scientific Research Centre Conferences, April 28-29, 2012

Singapore.

11. Ira Chaudhary, Faria Fatima, Smita Rastogi, Neelam Pathak. Isolation of

Ligninolytic Enzymes - producing Microorganisms from the Soil. National seminar

on reactive oxygen species: roles in animal and plant biology Dec 23 – 24.

12. Faria Fatima, Ira Chaudhary, Neelam Pathak. Biochemical and molecular analysis

of Rhizosphere microbe’s National symposium on biodiversity and food security:

Challenges and divising strategies. Dec 10- 11 IIPR Kanpur.

13. Ira Chaudhary, Faria Fatima, Smita Rastogi, Neelam Pathak. Biodiversity analysis

of lignin degrading microorganisms by assaying enzymes. National symposium on

biodiversity and food security: Challenges and divising strategies. Dec 10- 11 IIPR

Kanpur.

14. Faria Fatima, Ira Chaudhary, Jasarat Ali, Smita Rastogi and Neelam Pathak. Direct

isolation of DNA from soil and its PCR amplification for microbial diversity

analysis. SAARC Workshop on Biodiversity Conservation, Sep. 21- 22, 2010.

15. Ira Chaudhary, Faria Fatima, Smita Rastogi and Neelam Pathak. Isolation and

characterization of microbes with phosphate solubilizing and lignin degrading

abilities. SAARC Workshop on Biodiversity Conservation, Sep. 21- 22, 2010.

Poster Paper- (Total: 5)

1. Faria Fatima, Jiya Naseem, Erum Sheikh, Pawan Kushwaha, Preeti Bajpai, Neelam

Pathak. Extracellular synthesis of Silver and Gold nanoparticles by Bipolaris

tetramera and their antibacterial activity. Innovations in sciences for better tomorrow,

“LUSCON 2013” 20-21 March 2013.

2. Faria Fatima, Erum Sheikh, Pawan kushwaha, Prriyabka verma, Priti bajpai, Neelam

Pathak and A.K srivastava.Fungus mediated extracellular synthesis of silver and Gold

nanoparticles and their antibacteria activity. National seminar on stress, development

and adaptation: Biochemical Basis and Biotechnological Approaches 15-16 March

2013.

3. Faria Fatima, Nilesh Kumar, Sarika Singh, Preeti Bajpai and Neelam Pathak.

Biological Synthesis of Nanoparticles by Novel Phosphate solubilizing Fungus

(Bipolaris tetramera) and their antibacterial activity. Alumni Meet and National

Seminar on “Frontier Agriculture-2013”. Deva University Of Agriculture and

technology, Kumarganj, Faizabad April 20-21, 2013.

4. Faria Fatima, Ira Chaudhary, Neelam Pathak Smita Rastogi and Rolee Sharma.

Isolation, Biochemical and Molecular Characterization of microbes with phosphate

solubiling abilities. International Conference on Microbial, Plant and Animal research

(ICMPR-2012),March 29-31,2012.

5. Ira Chaudhary, Faria Fatima, Smita Rastogi, Neelam Pathak. Isolation of

Ligninolytic Enzymes producing Microorganisms from the Soil. National seminar on

reactive oxygen species: roles in animal and plant biology, Dec 23 - 24 2011.

Poster presentation Awards

First Best Poster presentation Award

Faria Fatima, Nilesh Kumar, Srika Singh ,Preeti Bajpai and Neelam

Pathak.Biological Synthesis of Nanoparticles by Novel Phosphate solubilizsing

Fungus (Bipolaris tetramera) and their antibacterial activity. Alumni Meet and

National Seminar on “Frontier Agriculture-2013”. Narendra Deva University Of

Agriculture and technology, Kumarganj Faizabad. April 20-21, 2013.

Second Best Poster Award

Faria Fatima, Jiya Naseem, Erum Sheikh, Pawan Kushwaha, Preeti Bajpai, Neelam

Pathak. Extracellular synthesis of Silver and Gold nanoparticles by Bipolaris

tetramera and their antibacterial activity. Innovations in sciences for better

tomorrow, “LUSCON 2013” 20-21 March 2013.

Oral Presentation for Young Scientist award in SAARC Workshop at BHU, Varanasi

Faria Fatima, Ira Chaudhary, Jasarat Ali, Smita Rastogi and Neelam Pathak. Direct

isolation of DNA from soil and its PCR amplification for microbial diversity analysis.

SAARC Workshop on Biodiversity Conservation, Sep. 21- 22, 2010.