science & technology option-1 - ijerstdistribution with depth also vary among soils. the...
TRANSCRIPT
200
This article can be downloaded from http://www.ijerst.com/currentissue.php
Int. J. Engg. Res. & Sci. & Tech. 2014 N W Bagalkar, 2014
EVOLUTION OF PHOSPHOBACTER
FROM SALINITY AFFECTED SOIL
IN AMARAVATI DISTRICT
N W Bagalkar1*
The salinity affected soil was investigated for the study of phosphate solubilization by bacteriaand fungi. In the present study 87 soil sample were collected from salinity affected area ofAmravati district (Daryapur, Bhatkuli, and Anjangaon), among these samples, 34 samples showedthe ability to solubilize the inorganic insoluble phosphate. From the study it was observed thatthe fungi (Aspergillus spp., Penicillium spp.) have more solubilizing ability of inorganic insolublephosphate than bacteria, i.e., B. cereus, B. megaterium, Bacillus subtilis, pseudomonas spp.,Enterobacter spp., Hence the application of biofertilizer prepared by above mentioned fungishould be helpful to increase the crop yield by solubilizing large concentration of inorganic insolublephosphate.
Keywords: Phosphate Solubilizing Bacteria (PSB), Pikovaskayas agar, Biofertilizer
*Corresponding Author: N W Bagalkar � [email protected]
INTRODUCTION
Microorganisms have the ability to solubilize the
insoluble phosphates and maintain the nutrient
status of soil (Richardson, 2001). Microorganisms
are central to the soil P cycle and play a significant
role in mediating the transfer of P between
different inorganic and organic soil P fractions,
subsequently releasing available P for plant
acquisition (McLaughlin et al., 1988 and Oberson
et al., 2001). Phosphorus (P) is one of the major
essential macronutrients for plant and is applied
to soil in the form of phosphatic fertilizers. In soil
inorganic and organic forms of phosphorus is
present. The inorganic forms of the element in
1 Department of Microbiology Sant Gadge Baba Amravati University, Amravati (INDIA).
Int. J. Engg. Res. & Sci. & Tech. 2014
ISSN 2319-5991 www.ijerst.com
Vol. 3, No. 2, May 2014
© 2014 IJERST. All Rights Reserved
Research Paper
soil are compound of calcium, iron, aluminum and
fluorine. The organic forms are compounds of
phytins, phospholipids and nucleic acid which
come mainly by way of decaying vegetation.
Therefore, soils containing high organic matter
are also rich in organic forms of phosphorus
(Subbarao, 1982).
Amravati district of Maharashtra state in India
is a part of alluvial valley of Purna basin. The
district lies between latitude 20o, 37’ and 21o, 26’
N and longitudes 76o, 37’ and 78o, 27’ E in
northeastern part of Maharashtra. The district
covers an area of about 12,212 sq/km, out of
which 3053 sq. km (25%) is covered by Purna
201
This article can be downloaded from http://www.ijerst.com/currentissue.php
Int. J. Engg. Res. & Sci. & Tech. 2014 N W Bagalkar, 2014
alluvium. The salinity-affected area is 1756 sq.
km. (58% of alluvium) and occurs along central
part of river basin, which is located in
northwestern part of the district. The salinity in
shallow as well as deep groundwater on one hand
and scarcity of surface storage due to low rainfall
on the other makes the situation worse for
management of water resources for both drinking
irrigation and industrial use.
A large portion of soluble inorganic phosphate
applied to the soil as chemical fertilizer is
immobilized rapidly and becomes unavailable to
plant (Gorstein, 1986). Microorganisms are
involved in a range of processes that affect the
transformation of soil phosphorous and are thus
an integral part of the soil phosphorous cycle. In
particular, soil microorganisms are effect in
releasing phosphorous form inorganic and
organic pools of total soil phosphorous through
solubilization and mineralization (Hilda and Fraga,
1999).
The average soil phosphorus concentration is
about 0.05 ppm and varies widely among soil.
The phosphorus concentration required by most
plants varies from 0.003 to 0.3 ppm and depends
on the crop species and level of production
maximum corn grain yield may be obtained with
0.01 ppm phosphorus, if the yield potential is low,
but 0.05 ppm phosphorus is needed under high
yield potential. Organic phosphorus represents
about 50% of total phosphorus in soil and typically
varies between 15 and 80% in most soils. Soil
organic phosphorus decrease with depth and the
distribution with depth also vary among soils. The
phosphorus content of soil ranges from about 1-
3%. Therefore, the quantity increases with
increasing organic C and N. However, the C: P,
N: P ratios are more variable among soils than
the C: N ratio, (Miller and Donahue, 1990).
Most of the Indian soil (98%) is poor in
availability phosphorus to plants (Gaur, 1987).
Agricultural soils generally contain adequate
amount of total phosphorus but the amount of
plant available phosphorous is small (Singh and
Kapoor, 1994). Therefore application of
phosphatic fertilizers is essential for optimum
crop yield. But the utilization efficiency of
phosphate fertilizers by plant is only 20-25%
largely due to its chemical fixation in soil (Dave et
al., 2003).
It is generally accepted that the mechanism
of mineral phosphate solubilization by Phosphate
Solubilizing Bacteria (PSB) strains is associated
with the release of low molecular weight organic
acids (Goldstein, 1995; Kim et al., 1997) like
formic acid, acetic acid, lactic acid, sulphuric acid
and propionic acid, which through their hydroxyl
and carboxyl group chelate the cations bound to
phosphate, thereby converting it into soluble
forms (Kpomblekou and Tabatabai, 1994).
However, P-solubilization is a complex
phenomenon, which depends on many factors
such as nutritional, physiological and growth
conditions of the culture (Reyes et al., 1999).
There is experimental evidence to support the role
of organic acids in minerals phosphate
solubilization (Haldar et al., 1990).
The present investigation is carried out to study
the performance of phosphate solubilizing
microorganism in salinity affected area if Amravati
district (Daryapur, Bhatkuli, and Anjangaon)
Though lot of work have been made on phosphate
solubilizing microorganism from different parts
of India and Maharashtra but not much work has
been done on phosphate solubilizing microorganism
or on report is available on study of phosphate
solubilizing microorganism in salinity affected
area of Amravati district (Figure 1).
202
This article can be downloaded from http://www.ijerst.com/currentissue.php
Int. J. Engg. Res. & Sci. & Tech. 2014 N W Bagalkar, 2014
The objective of this study was to isolate and
characterized PSB from the salinity affected area
in Daryapur, Bhatkuli, and Anjangaon.in Amravati
district. As chemical fertilizers and phosphate
solubilizing biofretilizer are used by farmer to
increase the production in present days, there is
necessity to have a comparative study on
potential of native phosphate solubilizing
microorganism, which will help to diagnose the
need of phosphate solubilizing biofertilizer
application in the Amravati district (Daryapur,
Bhatkuli, and Anjangaon).
MATERIALS AND METHODS
Sample Collection
Total 87 soil samples were collected from salinity
affected area of Amravati district (Daryapur,
Bhatkuli, and Anjangaon) in sterilized container.
The soil suspension was prepared by mixing 1 g
of soil sample in 9 mL distilled water then
supernatant was discarded and soil sample was
point inoculated on previously prepared and
sterilized pikovaskaya’s agar plates. Then the
pikovaskaya’s agar plates were incubated at 28±2oC for 24-48 h. And after completion of incubation
time, Zone of phosphate solubilization was
recorded. The colonies showing clear zone of
solubilization were further subculture on
pikovaskaya’s agar plates.
Microscopic Study of Bacteria
Size, shape, arrangement and gram’s nature of
the isolates were studied for gram’s staining.
Smear was prepared from the isolated culture
on clean glass slide, heat fixed and stained. The
stained smear was observed under microscope
(Oil immersion lance-100x). The fungal isolates
were identified up to generic level based on their
colony morphology and microscopic examination
as outlined in the manual of (Gilman, 1957)
Identification of Bacterial IsolatedThrough Biochemical Test
The PSBs isolated from salt affected soils were
identified up to generic level based on
Figure 1: Map of Study Area
203
This article can be downloaded from http://www.ijerst.com/currentissue.php
Int. J. Engg. Res. & Sci. & Tech. 2014 N W Bagalkar, 2014
morphological and biochemical tests as specified
in Bergey’s Manual of Determinative Bacteriology
(Holt et al., 1994). Biochemical test were
performed as suggested by (Garrity et al., 2001)
which included following tests Grams, IMViC
reaction, catalase test, starch hydrolysis test
oxidation fermentation test, Phenyl alanine
deamination test, Nitrate reduction, Gelatin
hydrolysis test, Urea hydrolysis test,
Dehydrogenase test, Casein hydrolysis test,
Citrate utilization test, Indol production test, Triple
Sugar Iron (TSI) test, Carbohydrate fermentation
test (Glucose, Fructose, Sucrose, Arabinose,
Mannitol , Lactose, Treholase, Galactose,
Raffinose), Motility test, Endospore staining and
capsule staining.
Phosphate Solubilization by Plate Assay
Solubilization of tricalcium phosphate was
detected in Pikovskaya’s Agar medium (Sundara-
Rao and Sinha., 1963) each isolate was point
inoculated in at the center of Pikovskaya’s Agar
plate and inoculated for 24 – 48 h the development
of clear around the colony indicated phosphate
solubilizing activity.
Observe the zone of solubilization and maser
diameter around the colony
RESULTS
On the basis of cultural character, Morphological
character and biochemical character phosphate
solubilizing bacteria was identifying. Following
character was compare with ‘BERGEY’S
MANUAL’ and all phosphate solubilizing bacteria
and fungi were identified. That is Bacillus cereus,
Bacillus megaterium, Bacillus subtilis,
pseudomonas spp., Micrococcus spp.,
Enterobacter spp., fungi (Aspergillus spp.,
Penicillium spp.)
Out of this isolate fungi (Aspergillus spp.,
Penicillium spp.) having efficiency of Phosphate
solubilization was more as compare to other
isolated phosphate solubilizing bacteria that is
(285). But Enterobacter spp. having efficiency of
Phosphate solubilization was less as compare
to other isolated phosphate solubilizing bacteria
that is (127). Efficiency of Phosphate solubilization
was determined by plate assay using
Pikovaskaya’s Agar Medium (Figure 2).
To Isolate Phosphate SolubilizingBacteria and Fungi from Amravati District(Daryapur, Bhatkuli, and Anjangaon)
• Colonies showing zone of clearance were
observed on Pikovaskaya‘s agar plates.
• The ability to solubilize precipitated phosphate
was positively exhibited by Pseudomonas spp.
Bacillus cereus, Bacillus megaterium, Bacillus
subtilis, Micrococcus spp., Enterobacter spp.,
fungi (Aspergillus spp., Penicillium spp.).
• All phosphate solubilizing bacteria and fungi
was selected and subculture on Pikovaskaya‘s
agar plates for further studies.
Determination of Efficiency of Phosphate
solubilization, solubilize by. Bacillus cereus,
Bacillus megaterium, Bacillus subtilis
pseudomonas spp., Micrococcus spp.
Enterobacter spp., fungi (Aspergillus spp.,
Penicillium spp.)
% of Efficiency of PSB was calculated by using
following formula
100lub ×
ColonyofDiameter
DiameterilizaionSo
Table 1 represents the percentage efficiency
of different PSB using Pseudomonas spp.
204
This article can be downloaded from http://www.ijerst.com/currentissue.php
Int. J. Engg. Res. & Sci. & Tech. 2014 N W Bagalkar, 2014
Table 1: Efficiency of Phosphate Solubilization
S. No. PSB Strain Colony Diameter Solubilization Diameter / % Efficiency 48 Hr
1 Pseudomonas spp. 0.9 1.6 177
2 Bacillus cereus 1 1.6 160
3 Pseudomonas spp. 0.8 1.4 175
4 Bacillus megaterium 1.1 1.8 163
5 Aspergillus spp 0.7 2 285
6 Penicillium spp 0.8 1.6 200
7 Enterobacter spp. 1.1 1.4 127
8 Bacillus subtilis 0.9 1.4 155
9 Micrococcus spp. 0.7 1.4 200
10 Fungi (N. I.) 0.8 1.8 225
11 Fungi (N. I.) 1.1 2 180
Figure 2: Result of Efficiency of Phosphate Solubilizer
205
This article can be downloaded from http://www.ijerst.com/currentissue.php
Int. J. Engg. Res. & Sci. & Tech. 2014 N W Bagalkar, 2014
Bacillus cereus, Bacillus megaterium, Bacillus
subtilis, pseudomonas spp., Micrococcus spp.,
Enterobacter spp., fungi (Aspergillus spp.,
Penicillium spp.)
DISCUSSION
Phosphate soluibililizing bacteria survive well in
the soils which provide organic substrates,
nutrient, minerals, adequate moisture and
tolerable environmental conditions. Efficient
phosphate solubilizer always prefers soils which
have good carbon for their survival.
From the salinity affected area of Amravati
district. Total 87 soil sample from three different
tahshil (Daryapur, Bhatkuli, and Anjangaon) were
collected and the different species of phosphate
solubilizing bacteria were isolated using
pikovaskaya’s media. Out of 87 soil samples, 34
containing the different species of Phosphate
solubilizing bacteria and fungi. Among these
isolated Phosphate solubilizing bacteria, only
Phosphate solubilizing bacteria were identified on
the basis of following tests Grams, IMViC reaction,
catalase test, starch hydrolysis test, oxidase test,
Phenyl alanine deamination test, Nitrate
reduction, Gelatin hydrolysis test, Urea hydrolysis
test, Dehydrogenase test, Casein hydrolysis test,
Citrate utilization test, Indol production test, Triple
sugar iron (TSI) test, Carbohydrate fermentation
test ( Glucose, Fructose, Sucrose, Arabinose,
Mannitol, Lactose, Treholase, Galactose,
Raffinose), Motility test, Endospore staining and
capsule staining.
Out of isolates 34 are bacteria and fungi in
which 23 are of genus Bacillus, 4 are of
pseudomonas spp. and remaining 7 are of fungi
which are fungi (Aspergillus spp., Penicillium
spp.) and other are not identified. Different species
of bacillus like Ba.cereus are 11, Ba.megaterium
are 7 and Ba.subtilis are 5 in number.
Among the bacteria the genus bacillus was
dominating than pseudomonas spp., among the
Ba.cereus is found to be the most dominating
followed by Ba.megaterium and Ba.subtilis. In
Daryapur tashil Ba.subtilis and fungi Aspergillus
spp. were most dominating than Ba.Megaterium
and Penicillium spp. whereas pseudomonas spp.
completely absent. In Anjangaon tehsil Penicillium
spp. and Ba.Megaterium is dominating followed
by pseudomonas spp. Ba.subtilis and Ba.cereus.
In Bhatkuli tehsil pseudomonas spp. is dominating
followed by Ba.Megaterium, Penicillium spp,
Aspergillus spp., Ba.subtilis and Ba.cereus.
Bilolkar et.al. (1996) reported that Bacillus spp.
was mostly dominating in the soil of Latur,
Osmanabad and Parbhani whereas
pseudomonas spp. was dominating in the soil
from Aurangabad to Nanded district.
Bhattacharya et.al. (1997) reported that
bacteria were found as the predominant
phosphate solubilizing microorganism in all
Vidarbha soil followed by fungi.
Production of enzyme like phosphates is other
mechanism of phosphate solubilization
(Rodriguez and Fraga, 1999). Aspergillus spp
isolates, showed high activity of AP at 11 M of P;
however, the production of this enzyme was under
detection limit in excess of phosphate compared
to limiting condition, which could explained that
the synthesis of alkaline phosphatase by these
bacteria was inducible in low Pi, while it was
repressed in high concentration. These results
are in concordance with solubilization activity
Pikovaskaya’s Agar, where Aspergillus spp.
isolates were strong P solubilizer. Interestingly,
Enterobacter spp. strain produced a smaller drop
206
This article can be downloaded from http://www.ijerst.com/currentissue.php
Int. J. Engg. Res. & Sci. & Tech. 2014 N W Bagalkar, 2014
in pH value compared to others isolates. This
might suggest that this strain is capable to
solubilize phosphate by other ways than the
production of organic acid. Therefore, we found
a positive correlation between phosphate-
solubilizing capacity and phosphatase enzyme
activity.
CONCLUSION
It is concluded from the present study that all
isolated phosphate solubilizing bacteria and fungi
from salinity affected soil in Amravati district
(Daryapur, Bhatkuli, and Anjangaon) are very
useful for increasing solubilization of inorganic
insoluble phosphate. This isolate are very
important for increasing crop yield which is taken
in salinity affected soil in productivity of, cotton,
jawar, bazri, soybean (Glycine Max) etc. From
the study it was observed that the fungi
(Aspergillus spp, Penicillium spp.) have the more
solubilizing ability of inorganic insoluble phosphate
than bacteria, i.e., B.cereus, B.megaterium,
pseudomonas spp, Enterobacter spp., Hence the
application of biofertilizer prepared by above
mentioned fungi should be helpful to increase the
crop yield in salinity affected soil by solubilizing
large concentration of inorganic insoluble
phosphate.
ACKNOWLEDGMENT
Author is very much thankful to Principal of J D P
S College Daryapur for allowing working in
laboratory and available all the facilities, Dr. N V
Phirke and Dr. Nikhil Bajaj for their useful guidance
throughout the period of work.
REFERENCES
1. Bhattacharya P, Ghosh T K and Jain R K
(1997), “Evaluation of native phosphate
solubilising microorganism from vidarbha
soil”, J. Maharashtra agric. Univ., Vol. 22,
No. 2, pp. 252-253.
2. Bilolikar M N, Raut R S, Malewar G U,
Rachewad S N and Lawand B T (1996),
“Occurence of phosphate solubilizing
bacteria in soil of Marathwada region”, PKV
Res. J., Vol. 20, No. 1, pp. 18-20.
3. Dave A and Patel H H (2003), “Impact of
different carbon and nitrogen sources by
pseudomonas fluorescence”, Ind. J.
Macrobiol., Vol. 43, No. 1, pp. 33-36.
4. Garrity G M, Winters M and Searles D
(2001), Bergey’s Manual of Systematic
Bacteriology, 2nd Edn., Springer-Verlag, New
York, 2001.
5. Gaur A C (1987), “Organic Manures and
Biofertizer” (div. of Microbiology, Agri.
Research Instituted, New Delhi), pp. 46 and
124.
6. Giluman J C (1957), A manual of soil fungi,
Ames. Iowa USA: Iowa State Univ. Press.
7. Glick B R (1995), “The enhancement of
plant growth by free-living bacteria”, Can. J.
Microbiol., Vol. 41, pp. 109-117.
8. Gorstein A H (1986), “Bacterial solubilization
of mineral phosphates: historical
perspectives and future prospects”, Am. J.
Altern. Agricult., Vol. 1, pp. 57-65.
9. Goldstein A H (1995), “Resent progress in
understanding the molecular genetics and
biochemistry of calcium phosphate
solubilization by Gram negative bacteria”,
Biol. Agric. Hort., Vol. 12, pp. 185-193.
10. Halder A K, Bhattacharya P and
Chakrabarthy P K (1990), “Solubilization of
207
This article can be downloaded from http://www.ijerst.com/currentissue.php
Int. J. Engg. Res. & Sci. & Tech. 2014 N W Bagalkar, 2014
rockphosphate by Bradyrhizobium”, J.Gen.
Appl. Microbial., Vol. 36, pp. 81-92.
11. Hilda R and Fraga R (1999), “Phosphate
solubilizing bacteria and their role in plant
growth promotion”, Biotechnol. Adv., Vol. 17,
pp. 319-359.
12. Holt J G, Krieg N R, Sneath P H A, Stanley J
T and William S T (1994), Bergey’s Manual
of Determinative Bacteriology, 9th Edition,
Lippincott Williams and Willikins, Baltimore,
Maryland, USA, p. 787.
13. Kim K Y, Jorden D and Krishnan H B (1997),
“Rahnella aqualitis, a bacterium isolated
from soybean rhizosphere, can solubilize
hydroxyapetite”, FESM Microbial. Lett., Vol.
153, pp. 273-277.
14. Kpoblekou K and Tabatabai M A (1994),
“Effect of organic acids on release of
phosphorous from phosphate rocks”, Soil
Sci., Vol. 158, pp. 442-453.
15. McLaughlin M J, Alston A M and Martin J K
(1988), “Phosphorus cycling in wheat-
pasture rotations. II. The role of the microbial
biomass in phosphorus cycling”, Australian
Journal of Soil Research, Vol. 26, pp. 333-
342.
16. Miller R W and Donahue R L (1990), “Soils
– An introduction to soil and plant growth”,
Sixth Edition, Prentice Hall Publications.
17. Oberson A, Friesen D K, Rao I M, Bühler S
and Frossard E (2001), “Phosphorus
transformations in an oxisol under
contrasting land-use systems: The role of
the microbial biomass”, Plant and Soil Vol.
237, pp. 197-210.
18. Reyes I, Bernier L, Simard R and Antoun H
(1999), “Effect of nitrogen source on
solubilization of different inorganic
phosphates by an isolate of Penicillium
rugulosum and two UV-induced mutants”,
FEMS Microbial. Ecol., Vol. 28, pp. 281-290.
19. Richardson A E (2001), “Prospects for using
soil microorganism to improve the
acquisition of phosphorus by plants”, Aust.
J. Plant Physiol., Vol. 28, 897D906.
20. Rodriguez H and Fraga R (1999),
“Phosphate solubilizing bacteria and their
role in plant growth promotion”, Biotechnol
Adv., Vol. 17, pp. 319-339.
21. Singh S and Kapoor K K (1994),
“Solubilization of insoluble phosphate by
bacteria isolated from different sources”,
Environment and Ecology, Vol. 12, No. 1,
pp. 51-55.
22. Subbarao N S (1982), Advances in
Agriculture Microbiology, Oxford & IBH, New
Delhi, p. 296.
23. Subbarao N S (1982), Biofertilizer in
Agriculture and Forestry, Third Edition, pp.
129-135.