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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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To Isolate Phosphate Solubilizing Microorganism
#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.
2
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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
11
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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
14
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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 $urholderia, alstonia species and
15
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Pantoea. The identification of bacterial colonies too place by analyzing their r1/* with the
sequencing analysis. The occurrence of the very small amount of gluconic acid was found which
proved the immobilization of phosphorus at a lower pace. The study suggested that when the soil
is enriched with minerals lie 5e then fi'ation process taes place and the activity of PS$ 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
23
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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
26
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
27
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
28
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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.