To Isolate Phosphate Solubilizing Microorganism

7/25/2019 To Isolate Phosphate Solubilizing Microorganism

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To Isolate Phosphate Solubilizing Microorganism

Word Count: 7500

[ame o! the Writer"

[ame o! the Institution"


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#bstract

Phosphorus has been acquired by the plants in the soluble form which is found in the soil in the

minimum quantity. The available phosphorus is found in the soil, which reacts with the other

minerals available in the soil lie calcium, iron and aluminium causing the precipitation of the

compounds leaves the phosphorus in the insoluble form. The role of phosphorus!solubilizing!

bacteria are found to be very important in the conversion and decomposition in order to produce

a soluble form of phosphorus to the plants so that it can be utilized for plants" growth and

development. Moreover, the observable and gradual decrease in the p# indicates the activity of

PS$ for phosphorus immobilization by showing the traces of inorganic acids. The current study

aimed to isolate the phosphate solubilizing bacteria by analyzing the four soil samples, i.e.

banana, ground, garden and graveyard soil and analyzing the rhizosphere under the P%& mediumand tricalcium phosphate. The halozone colonies indicated the presence of Pseudomonas

fluorescens and the chemical reaction indicated the production of organic acids and the

e'periment indicate the immobilization of phosphorus.

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Contents

(ist of Tables..................................................................................................................2

)#*PT+ - I/T012)TI0/......................................................................................... 2

)#*PT+ 3 (IT+*T2+ +&I+4.................................................................................2

3.- Isolation of Strains................................................................................................... 2

3.3 5ormation of Phosphate!Solubilizing!$acteria 6 PS$........................................................2

3.7 Phosphorus Solubilizing Mechanism.............................................................................2

)#*PT+ 7 M+T#010(089........................................................................................2

7.- Sample.................................................................................................................. 27.3 Preparation of medium..............................................................................................2

7.7 Isolation of PSM......................................................................................................2

7.: Morphological )haracterization...................................................................................2

7.; 8ram Staining.........................................................................................................2

)#*PT+ : +S2(TS */1 1IS)2SSI0/.........................................................................2

:.- Isolation of PS$......................................................................................................2

)#*PT+ ; )0/)(2SI0/............................................................................................ 2

eferences..................................................................................................................... 2

3


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$ist o! Tables

Sr% o Title Page o%

- Table - 6 Prepartion of Medium 3:

4


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CPT'( ): IT(*+,CTI*

Plants containing adequate amounts of phosphorus entail appropriate growth and development.

Phosphorus plays a vital role by being a component of plant structure which is responsible to

regulate the protein synthesis mechanism in plants. The mechanism of cell division, distribution

and transformation of energy in terms of glucose taes place under the sufficient presence of

phosphorus in plants. Moreover, the process of tissue development is also buffered paving way

for the proper growth in plants. 0n the contrary, the deficiency of phosphorus can cause revert

the growth mechanism in plants while changing the normal color of leaves to dar!green color or

brown and mae the plant vulnerable ?.

The detailed wor of #olford


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stage, production of fruit and increased production of crops. Phosphorus plays a vital role in the

photosynthesis process, providing the essential carbohydrates to the plant. The phosphorus is

assimilated to plant, particularly, as anionbut the process of utilization of phosphorus taes place

slowly, which in turn, becomes essentially required to mae sure that phosphorus is available

adequately.

The deficiency of phosphorus in plants leads to the adverse effects on the growth and

development of plants. Moreover, insoluble phosphorus that has been already present in soil and

fertilizers can be immersed so that the absorption process of phosphorus can tae place

efficientlyand this available solution of phosphorus can convert in the soluble minerals essential

for the plants. This process taes place when the microorganisms i.e. $acteria or fungi

decompose the available insoluble phosphorus in soluble phosphorus and the process is nown

as immobilization. The current study aimed to isolate the microorganisms that play vital role in

the solubilizing of phosphate to ensure the ma'imum uptae of phosphorus in the plants.

CPT'( -: $IT'(#T,(' ('.I'W

-%) Isolation o! Strains

The comprehensive wor of 5rageria


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phosphorus in the form of hydrogen phosphate in rich soil where aluminium prevails and

reaction between these minerals causes the precipitation of phosphorus ions on *luminium.

These minerals are available in the soil which causes the fi'ation of phosphorus in the form of

hydrogen sulfate and precipitates of *luminium. The chemical equation for the process is as

follows

*l7DD #3P0:!


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)a


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Therefore, the study concluded by postulating thatby using PSM in agricultural processes can be

beneficial to increase the crop yields by the activity of PSM the availability to phosphorus to the

plant increases which leads to better agricultural yields.

The brief wor of Pradhan and Sula


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-%- /ormation o! PhosphateSolubilizing1acteria 2 PS1

*ccording to )habot, et al. ?, the production of crops can be increased by maing the

plants absorb the proper amount of phosphorous, which stimulate the e'tent to which the plants

grow. It has also been found that when the occurrence of rhizobacteria taes place across the

plant roots, then they also stimulate the absorption of phosphorus in plants. It is required that

there should be the dense colonization of PS$ in order to sustain the absorption of phosphorus is

the plants at the ma'imum level. Therefore, the broader the rhizosphere the more effective the

absorption of phosphorus will tae place. Moreover, it is also necessary that there e'ists

appropriate level of minerals and nutrients to ensure the proper benefits of the colonization of

rhizobacteria or P8P. The current study uses the sample of rich and poor soil consisting of the

te'ture of silty!clay and silty loam under the presence of rhizobia PSM leguminosarum to

e'amine the growth of maize. The temperature was 3; centigrade under the greenhouse where

si' seeds were disseminated to analyze their growth rate by adding the fertilizer /!/# :/07, %!

%)l and P!superphosphate. The results from the conFugated strains of l(u'Dindicated that the

plant growth, i.e. maize growth was not affected by the fertilization of soil with phosphorus in

poor soil as well as the colonization of bacteria PSM was also not affected by the procedure.

Therefore, the study postulates that plant growth can be enhanced by the solubility of

phosphorus, but the procedure is effected only in the presence of rich soil.

&asquez, et al.


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insoluble phosphorus to soluble phosphorus taes place by the production of some organic acids

by the fungi. These fungi or bacteria are surrounded by the rhizoshpere which stimulates the

nitrogen fi'ation process and increase the phosphorus intae of the plants resulting in the

enhanced growth and production of plants or crops. In the current study, an e'periment

consisting of sample soil cores, which was found to be enriched with calcium, aluminium and

iron was e'amined and two species of mangrove plants were harvested and the microorganisms

from the soil were studied. The insoluble phosphorus was present higher in quantity than soluble

phosphorus. The results from the culture study indicated the presence of phosphorus in mangrove

roots and $acillus licheniformis was found to be accountable for the absorption of phosphorus.

The solubility of phosphorus was found to be enhanced when the fungal colony was e'posed to

*. /iger indicating the direct correlated effect between them. 5urthermore, all types of bacteria

produced some organic acids to solubilize the insoluble phosphorus and these organic acids were

identified as acetic acids, lactic and isobutyric acid which helped in the immobilization of

phosphorus.

The study conducted by 0liveira, et al. ? aims to e'amine the microorganisms in order to

evaluate their activity related to the conversion of insoluble phosphorus. It has been inferred that

microorganisms play a vital role in the development of plants by providing them soluble and

absorbable minerals for their growth. The study conducted e'periment on the soil samples which

were e'tracted from the rhizosphere of maize and four samples were gathered which possessed

noticeable difference in phosphorous nutrient. The colonies of microorganisms around the

rhizosphere were e'amined which consisted of :; in number and were selected on the basis of

their efficiency to convert phosphorus and the process too place under the culture which

consisted of phytic acid i.e. )C#->03:PC, soybean lecithin, tricalcium phosphate and aluminium

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phosphate. The microbes consisting of bacteria were isolated by identifying the presence on

nucleotide in their r1/* whereas fungi were isolated by e'amining their internal transcribed

spacer that was present in their r1/*. The conversion of phosphorus was found to be highest in

bacterium mainly nows as $acillus species and $urholderia species and was their rate of

immobilization of phosphorus was measured which was found at the rate of C@G out of the total

amount of phosphorus from the compound of tricalcium phosphate. It has been observed that the

solubility of microorganisms and the immobilization of phosphorus highly depend on the nature

of the phosphorus compound with which the soil is enriched and on the microorganisms that are

involved in the process of phosphorus fi'ation.

*ccording to )hen, et al.


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found for the very first time in the isolation of phosphorus solubilization and proved their part by

secreting enzymes and converting the phosphorus. Therefore, the importance of PSM bacteria in

order to increase the phosphorus intae in plants and reduce the pollution by decreasing the use

of phosphate fertilizers has taen the boundless importance in the modern era.

The study conducted by ashid, et al.


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they are encountered with herbicides mainly consisting of o'adiazon and o'yfluorfen. 0n the

contrary, some herbicides have proven their negative effects on the activities of microorganisms

resulting in the harmful effects on plants. The e'periment too place by using the nutrients"

enriched soil in which rice seeds were sown and the rhizosphere sample was collected from

there. These soil samples were collected after removing the roots of the seedlings for CB days.

The diluted mi'ture of soil and distilled water was mi'ed with tricalcium phosphate, which

indicated the presence of microbes" colonies. *fter that, sodium bicarbonate was added in order

to e'amine the rhizosphere. The results indicated that the presence of herbicides has fastened the

activities of PSM and the phenomena was rationalized with the indication that the

microorganisms used these herbicides to get energy and hence promoted the decomposition of

insoluble phosphorus nutrients in the soil. 5urthermore, the number of microorganisms was

increased day by day with the presence of herbicides in the soil and the crop was also effected

with this activity by reaching at the stage of flowering by the 7Bthday. Therefore, the overall

study indicated that the presence of herbicides stimulated the PSM activity and growth, which

resulted in the greatest amount of phosphorus solubilization and hence, the ma'imum amount of

phosphorus became available for the plants resulting in enhanced growth and development of

crops.

#ameeda, et al. ? argued that the soil may be enriched with minerals and contain higher

amount of phosphorus, but their availability is not considered to be adequate for the plant uptae

in order to utilize it for the growth and development and it happens due to the availability of

other minerals available in the soil specifically calcium which reacts with the phosphorus and the

fi'ation process taes place in which the insoluble form of phosphorus is generated. The

fertilizers that are applied to the soil usually convert into the insoluble form of phosphorus

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following the fi'ation process and very small amount of phosphorus is absorbed into the plants

which prove to be quite inadequate for the proper growth of plants. Therefore, the necessity of

PSM is originated because these PSM are able to solubilize the insoluble form oh phosphorus

maing it available for the plants. The study conducted an e'periment in glasshouse by sowing

the maize seeds which were provided with nutrients for ten days and peat consisted of

microorganisms i.e. marcescens and Pseudomonas were also inserted and at the end, rhizosphere

was collected. The results indicated that the presence of PS$ produced organic acids, i.e.

gluconic acid, which indicated the decomposition process of insoluble phosphorus and the

observed growth in the length of shoot, root enhancement and decreased vulnerability of crops

was found. The study did not provide any result for the increase in the PS$ during the process

indicating that the number of PS$ included in the peat remained same throughout the process.

The study concluded that the use of marcescens and Pseudomonas during the incubation of seeds

will enhance the phosphorus solubilization process and will increase the crop yield.

The comprehensive wor of Perez, +. G to CBG ferrous o'ides and the amount of

phosphorus indicated to be very low. The soil samples were sterilized and were diluted with the

media nown as /$IP and tricalcium!sulphate. The appearance of halozone too place. 0n the

contrary, no traces of iron phosphate were found in the e'periment. The isolates from the

halozone were identified to have the bacterial colonies of $urholderia, alstonia species and

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Pantoea. The identification of bacterial colonies too place by analyzing their r1/* with the

sequencing analysis. The occurrence of the very small amount of gluconic acid was found which

proved the immobilization of phosphorus at a lower pace. The study suggested that when the soil

is enriched with minerals lie 5e then fi'ation process taes place and the activity of PS$ proves

to be less effective than the soil where 5e is found in lesser proportion. 5urthermore, the bacteria

strains which were used in this e'periment were proven to wor effectively under the presence of

aluminium. Therefore, it has been highly advised that the soil should be e'amined properly and

only such bacterium should be used for the immobilization process that are found to be efficient

in the conversion of phosphorus.

The significance of phosphorus in the plant growth has been discussed earlier and it has been

described that the absorption of phosphorus taes place in the soil in the soluble form. The

absorbed phosphorus reacts with the minerals available in the soil, i.e. aluminium, calcium and

ferrous which converts the phosphorus into the insoluble form by the fi'ation process. In order to

mae phosphorus available to the plants the phosphobacteria play a vital role in which they

decompose the inorganic form of phosphorus while releasing organic acids as a by!product. The

e'periment conducted by Ponmurugan and 8opi


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presence of phytohormones. The statistical analysis showed the positive relation between the

capacity of solubilizing phosphorus and the formation of phytohormones. The study concluded

that with the help of phosphobacteria the growth of the plants can be increased due to the

provision of adequate amount of phosphorus to the plants.

The study conducted by $abana and *ntoun


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the mechanism of PS$. The seeds of wheat were sown in the soil characterized as sandy loam

and were applied with the phosphorus!fertilizers in order to enrich the soil with phosphorus. The

preparation for the insertion of PS$ too place by using the P&% medium. 4hen plants were in

the growing stage, the samples of soil were gathered for the analysis of phosphorus absorption.

The results of the study indicated that the growth of plants was stimulated due to the adequate

absorption of phosphorus. The plant development indicated the better absorption of water and

nutrient by measuring the length of root and shoot. 4hen the PS$ containing hizobium was

doubled in the soil, then the growth of the plant again found to be at a greater pace than the

single dosage of PS$. Therefore, the study postulated and favored the previous studies with

respect to the positive correlation of phosphorus!fertilizers, PS$ hizobium and the overall yield

of the crop.

-%3 Phosphorus Solubilizing Mechanism

*ccording to #olford


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for the plants. The study reveals the process of phosphorus fi'ation and describes its importance

which proves to be viable for the proper growth and development of plants and its deficiency

proves harmful effects on the plants by maing them wea and producing abnormal leaf color

indicating the over processing of photosynthesis.

Silcoc, et al.


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phosphorus and face adverse effects. The phosphorus may be available in the soil, but plants fail

to absorb it because of its close bonding with minerals calcium, aluminium or iron. The activity

of PSM taes place in order to convert insoluble phosphorus to soluble phosphorus so that plant

can absorb and utilize the adequate amount of phosphorous essential for their growth. The

e'periment in this study too place with the four samples of same soil rice straw was sown and

Pseudomonas was added and the process was stimulated by adding glucose in order for the

formation of soil. The result of the study indicated that the soil was enriched after the phosphorus

fi'ation process, but this effect was not too significant whereas the significant growth in the plant

was observed. The study encompassed that modification of the soil contents in terms of

microorganisms can enhance the process of phosphorus fi'ation by motivating the

microorganisms that were already present in the soil even before the assimilation of bio!

fertilizers.

*lam, et al.


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analyzed for seven days. The results of the study indicated the presence of holozone colony

which proved the presence of the isolates of PSM out of which fungi proved to be most

solubilizing in nature for phosphorus. The continuous drop in p# also proved the production of

organic acids by the PSM consisting mainly of o'alic, acetic and citric acids. The results for the

immobilization of phosphorus proved that the bacteria were actively responsible for the

immobilization and the result was rationalized on the basis that fungi colonies are larger in size

than bacteria colonies which may result in higher immobilization by fungi. The study also

suggested that the production of organic acids play important role in the immobilization

mechanism.

Igual, et al.


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PS$ and proper characterization of soil, it is possible to provide specific bacteria that can mae

the phosphorus absorption process more efficient and can cause increased productivity of crops.

The comprehensive wor of Sahin et, al.


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early fruit ripening and flower formation, which as a whole, indicate the effective and efficient

growth. The phosphorus is assimilated from the soil to the plants in the form of phosphate anion

and this absorption is found to be at low paced which increase need to ensure the availability of

phosphorus in the soil. The lac of phosphorus in plants will cause the negative effects on the

growth and development of plants. Moreover, all the types of soil contain insoluble or inorganic

amount of phosphorus where fertilizers can be included in order to increase the amount of

phosphorus available to the plant and this process will convert the soluble form of phosphorus

which is essential for the plants. The study conducted by Mehrvarz H )haichi ? too place

an e'periment in the research field. The soil was characterized as clay loam and the soil was

applied with the fertilizer nown as triple!supper!phosphate. Pseudomonas was added in the soil

and the barley seeds were sown. These bacteria played vital role in the decomposing of the

inorganic phosphorus of organic phosphorus in order to mae it available for the plants. These

bacteria produced by!products in terms of organic acids ensuring the process of immobilization

has been taen place. The soil samples were collected, sterilized and were diluted with the media

nown as %&P media and tricalcium!sulphate. The appearance of halozone too place. The

occurrence of very small amounts of gluconic acid was found which proved the immobilization

of phosphorus at a lower pace. The study suggested that when the soil is enriched with

phosphorus and PS$ then fi'ation process taes place. Therefore, it has been concluded that the

application of PS$ inoculants should be practiced in order to increase the overall crop yield and

cost effectiveness

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CPT'( 3: M'T&*+*$*4

3%) Sample

The soil samples were collected from neighboring places consisting of banana soil, graveyard

soil, garden soil and ground soil and the samples were gathered from the site of cultivating areas.

The samples for each soil were taen C times from different sites at the depth of -@ inches. These

samples were dried and then all C samples were mi'ed together by maing one sample for each

type of soil.

3%- Preparation o! medium

Piovasaya agar or P%& agar was used as a medium to isolate PSM which is specifically used

for the detection of PS$. The p# of the medium was sustained at @. The components of the P%&

agar and their quantity used in the process is shown in Table -.

Composition 4ram6ml

8lucose -B

9east +'tract B.;


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water


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presence of gram negative bacteria, whereas the observation of purple colonies represented the

presence of gram positive bacteria.

CPT'( : ('S,$TS #+ +ISC,SSI*

%) Isolation o! PS1

The halozone of phosphate solubilization was found in three soil samples, indicating that the

proficiency of phosphorus solubilization has been generated.

These halozones were obtained from the medium of banana

soil sample which showed the dilution of -BK!7. Therefore, the

banana soil was found to be

proficient for the

immobilization of phosphorus

indicating the presence of

bacteria that are responsible

for the solubilization of phosphorus i.e. PS$. The halozone in banana soil can be seen in 5igure

-.

The result obtained from the halozone of the garden soil sample proved its proficiency of the

immobilization of phosphorus by indicating the e'istence of phosphorus!solubiling!bacteria and

the garden soil demonstrated the dilution of -BK!

:

. The halozone is shown in figure 3.

The third soil sample consisting of graveyard soil also showed the positive result by having the

ability to immobilize phosphorus, which in turn indicated the presence of PS$ and the banana

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5igure. 3 6 #alozone in

8arden Soil

5igure. - 6 #alozone in

$anana Soil


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soil sample e'hibited is dilution of -BK!:. There were no

isolates formed in the ground soil sample. The halozone in

graveyard soil are shown in figure 7.

The results indicated the presence of gram negative bacteria

due to its features which were containing the colorless

colonies with the absence of any pigmentE the cells were

found to be gram negative with rod!lie structure. Moreover, the biochemical reactions indicated

the presence of Pseudomonas fluorescens which were found during the process of gram staining.

0n the contrary, the isolates indicating the presence of gram positive bacteria due to its features

containing the white colonies with slimy shape and margins among them was found to be

irregular. These colonies possessed the e'istence of $acillus megaterium which was proved by

the biochemical reactions during the gram staining. Therefore, the presence of Pseudomonas and

megaterium indicated that the immobilization mechanism for the conversion of insoluble

phosphorus to the soluble phosphorus too place.

CPT'( 5: C*C$,SI*

The study conducted an e'periment by analyzing the four soil samples of banana, graveyard,

garden and ground soil in order to isolate the microorganisms that isolate phosphorus. The results

of the present study indicated that the three soil samples, i.e. 8arden, graveyard and banana soil

in which the isolates were formed in the samples from garden soil, graveyard soil and banana

soil. These samples indicated the formulation of halozone which represented the

microorganisms" colony lying under the P&% medium. These isolates were studied under the

microscope, which confirmed the e'istence of bacteria which are responsible to convert

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5igure. 7 6 #alazone in

8raveyard Soil


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phosphorus to soluble forms. The current study has declared the presence of bacteria, i.e.

Pseudomonas by which the immobilization of phosphorus too place. The absorption of

phosphorus is considered to be essential for plant development and has found to be the primary

nutrient for the plant growth and development. Microorganisms lie bacillus margarita and

Pseudomonas brea down inorganic or insoluble form of phosphorus and convert it into the

soluble or organic form so that phosphorus can be utilized by plants. This immobilization of

phosphorus mechanisms causes the secretion of organic acid, which confirms the activity of PS$

mechanism. The organic acid secreted as by!product from the bacteria cause the decrease in the

p# of soil, which proves the microorganism activity for the conversion of phosphorus.

In agricultural soil, inorganic form of phosphorus has already been e'isted, but the phosphorus

tends to be insoluble and becomes useless for the plant. The phosphorus added to the soil in the

form of phosphorus!fertilizers can increase the intae of phosphorus to the plants for a while, but

there e'ists other minerals in the soil lie calcium, aluminium, magnesium and iron which start

precipitating with the soluble phosphorus and turns it into the insoluble form or inorganic form.

In this way, phosphorus becomes unavailable to the soil and the plants face adverse effects of by

being deficient in phosphorus. Moreover, the absence of soluble phosphorus from the soil will

cause the increased demand of soluble phosphorus and if more phosphorus!fertilizers are applied

then the cost for the crop yield will be increased. Therefore, PS$ are required for the continuous

decomposition of insoluble phosphorus or inorganic phosphorus so that the plants will

continuously get the required amount of phosphorus and the hazardous effects of phosphorus

deficiency can be eliminated and plants can uptae all the essential nutrients properly.

In this way, the bacteria that brea down the inorganic form of phosphorus can be used along

with the bio!fertilizers which will ultimately increase the phosphorous efficiency in soil by

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breaing down the insoluble phosphorus to the soluble phosphorus and that will improve the

plant nutrient intae and will increase the crop yield. 5urthermore, when these bacteria or PSM

will be used as bio!fertilizers then not only the availability of phosphorus will be increased, but

the usage of phosphorus fertilizers will be reduced, which will play an important part in reducing

the environmental pollution and the overall agricultural and crop yield will be increased.

+ffective provision of phosphorus to the soil will result in the proper growth and development of

the plant with stronger root and shoot structure, increased productivity of fruits and flowering,

reduced vulnerability towards diseases and weathering. Therefore, phosphorus not only proves to

be essential for the plant as a nutrient, but it has also become important to be used in order to

increase the agricultural capacity.

(e!erences

Schachtman, 1. P. eid, . L. H *yling, S. M., ?. Phosphorus uptae by plants from soil to

cell.American Society of Plants Physiologists, 116


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ichardson, *. +. H Simpson, . L.,


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)hen, 9. P. eha, P. 1. *run, *.$. Shen, 5. T. (ai, 4. *. H 9oung, ). ).,


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httpOOwww.sciencedirect.comOscienceOarticleOpiiOSB=::;B-7BCBBB;>= *ccessed 3C

Lanuary 3B-;J.

Igual, L. &alverde, *. )ervantes, +. H &elazquez, +.,


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Mehrvarz, S. )haichi, M. . H *lihani, #. *., ?. +ffect of Phosphate solubilizing

microorganisms and phosphorus chemical fertilizer on forage and grain quality of barely

;;!>CB.

To Isolate Phosphate Solubilizing Microorganism

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