Volume 6, Issue 9, September – 2021 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

Isolation and Characterization of Phosphate

Solubilizing Microorganism (PSM) from the
Rhizosphere and Roots of Crops Indigenous to
Ihiagwa-Owerri Imo State Nigeria
Onyewenjo, S. C.1, Njoku, H. O.2 and Ire, F. S.2
1 Biology/Microbiology Department, Federal Polytechnic Nekede, Owerri, Imo State.
2 Microbiology Department, University of Port Harcourt, Port Harcourt, Rivers State.

Abstract:- The expensive eco-degrading chemical 2010). There abound in nature, wide range of
fertilizer demands an affordable sustainable eco-friendly microorganisms that can solubilize insoluble phosphate.
alternative: biofertilizer. Isolates from the rhizosphere These microbial solubilizers use several mechanisms which
and root samples of Abelmoschus esculentus, Manihot are necessary to maintain the global cycle (Whitelaw, 2000).
esculenta, Musa paradisiaca and Zea mays were obtained These unavailable forms of phosphates are solubilized by
using standard microbiological procedures, screened for microbes through excretion of organic acids and or
phosphate solubilization using Pikovkaya’s medium and production of phosphatase enzyme (Kucey, 1983). A large
their phosphate solubilization index(PSI), and efficiency number of bacteria including species of Pseudomonas,
determined. Isolates with PSI ≥ 140 were subjected to Azospirillum, Azotobacter, Klebsiella, Enterobacter,
morphological and biochemical characterization. Alcaligenes, Arthrobacter, Burkholderia, Bacillus,
Discrete colonies obtained were 145. Total bacterial Rhizobium, and Serratia have been reported to enhance
isolates were 111 and 34 fungal. Phosphate solubilizing plant growth with their different plant growth-promoting
isolates were 39 (26.8%), phosphate solubilizing index activities including phosphate solubilization. Phosphate
(PS1) range from 109.1 to 190. Isolates with PSI ≥140 solubilizing microbes can be used to ensure sustainable
were identified as Pseudomonas sp., organic farming systems and reduce the utilization of
Bacillus sp., Aspergillus sp., and Penicillium sp. The agrochemicals in agricultural fields (Widawati, 2011).
solubilization efficiency order of the isolates was
Pseudomonas> Bacillus> Pencillium> Aspergillus. The The work aims to isolate and characterize phosphate
application of these biofertilizer-producing solubilizing microbes from the rhizosphere and roots of
microorganisms will deliver an adequate amount of indigenous crops to Ihiagwa-Owerri Imo State Nigeria.
phosphorus plants thereby boosting agro development.
II. MATERIALS AND METHODS
Keywords:- Biofertilizer, Eco-Friendly, Phosphate
Solubilizing Index, Chemical Fertilizer, Phosphate 2.1 Sampling Collection
Solubilization Efficiency. Forty rhizospheric soil and 40 roots were sourced from
farmlands located at Ihiagwa-Owerri,
I. INTRODUCTION latitude 5o25’26” N, longitude 7o1’31” E. Ten
rhizospheric samples (soil and root) were sourced from an
Phosphorus (P) is among the three major plant assortment of Abelmoschus esculentus(okro), Manihot
nutrients required in the optimum amount for proper plant esculenta(cassava), Musa paradisiaca (plantain), and Zea
growth. A shortfall in the adequate P imparts negatively on mays(maize) plants. The samples were aseptically dug out
the yield of crops below the maximum economic level. from the depths 10-30 cm into sterile polythene bags after
Phosphorus’s roles in the metabolism of plants are the crests of the soils were cleared of debris with a clean
indispensable. Its biochemical roles extent to sterile trowel (Philippot et al., 2012). The samples were
photosynthesis, respiration, energy storage and transfer, cell transported to the laboratory at 40C temperature.
division, cell enlargement, and several other processes in the
living plant. An adequate supply of phosphorus in the early 2.2 Sample Preparation and Microbial Isolation
stages of plant growth is very essential. It promotes Composite sample from ten soil samples of each plant
physiological functions including early root formation and is type was subjected to ten-fold serial dilution and an aliquot
important for the development of the reproductive parts of (0.1ml) of dilution 10-2 was each spread plated on nutrient
plants. It is very necessary for seed formation. More agar (NA) and sabrourand dextrose agar (SDA) plates and
phosphorus is found in plants’ seeds than found in any other incubated at 30 ± 2oC for 2-7days. Discrete colonies were
part of the plant. It helps plants to survive stressors like stored in slants for further studies (Philippot et al., 2012).
winter rigors and some plants’ diseases (Balamurugan et al.,

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Volume 6, Issue 9, September – 2021 International Journal of Innovative Science and Research Technology
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Root samples were surface sterilized, macerated, and aided by hand magnifying glass), gram staining, and
subjected to ten-fold serial dilution, inoculation, and biochemical test (citrate utilization, catalase, urease, indole,
incubation as the soil samples (Philippot et al., 2012). methyl red, vogues Proskauer, H2S, sugar fermentation, and
nitrate reduction test) for bacteria; and cultural and
2.3 Screening of phosphate solubilizing microorganisms microscopic characteristics for fungi. The outcome was
(PSM) matched against Bergey’s Manual, 9th edition, and atlas of
Pikovskaya’s agar (PVK) {(grams/liter); Glucose, fungi (Behzadi and Behzadi, 2012, Cheesbrough, 2000, and
10.0gm; Ca3(PO4)2, 5.0 gm; (NH4)4SO4, 0.5 gm; NaCl, Willey et al., 2017).
0.2 gm; MgSO4.7H2O, 0.1gm, agar, 15g, pH 7.3±2}was
prepared (Oliveira et al., 2009), sterilization with an III. RESULTS
autoclaved and poured into sterile Petri dishes. Loopful of
48 hours old bacterial isolates and 3 days needle scrap of 3.1 Isolates Zone of Phosphate Solubilization
fungal isolates were centrally spot inoculated on sterile PVK The result of the phosphate solubilization test is
agar plates and incubated at 30±2oC (Prajapati and Modi, presented in Table 1. The diameter of the isolates’ zone of
2012). Colonies showing halo zones were taken as evidence clearance on Pikovskaya’s agar medium was measured in
of P-solubilization. The isolates with a clear halo zone were millimeters. The symbols (+) was employed to represent the
purified three times on PVK solid medium. The purified diameter of clearance ranging from 0-1.5mm, (++) represent
bacterial isolates were maintained on nutrient agar slant and the diameter of clearing ranging from 1.5-3.0 mm, and
fungal isolates on SDA slant. (+++) represent the diameter of the zone of clearing ranging
from 3.0-4.5 mm while above 4.5mm diameter is
2.4 Determination of Phosphate Solubilization Index represented by four pluses (++++). These measured the
(PSI) various abilities of the isolates to solubilize the insoluble
Qualitative estimation of P-solubilization was done by tricalcium phosphate component of the medium. The
measuring the PSI. Loopful of each isolate (48hours for solubilization was the reason for the clearance. Isolates that
bacterial and 3 days fungal) was spotted on the PVK solid could not solubilize mica, do not produce a zone of
medium and incubated at 30±2 °C for 5-7 days in three clearance. They are represented by a negative (-) symbol. A
replicates, with the sterile medium serving as a control. PSI total of 145 discrete isolates were obtained from the eighty
formula, PSI = C+H/C, (C = Colony diameter; H =Halo samples (forty soil samples, forty root samples). Thirty-nine
zone diameter) (Pathak et al., 2017). Isolates with PSI ≥140 (26.9%) were phosphate solubilizers. Out of the 39 isolates
were preserved for preliminary identification and further able to solubilize phosphate, 18(46.1%) solubilization
studies. ability was represented by one (+), 12(30.8%) solubilization
ability was represented by two (++), 6(15.4%) was
2.5 Preliminary Identification of PSM represented by three (+++) and 3(7.7%) was represented by
Preliminary identification was carried using colonial four (++++).
morphology (the colony colour, colony shape, and elevation

TABLE 1: Isolates Zone of Phosphate Solubilization

ZONE OF SOLUBILIZATION ISOLATES
PRZS -12, ORZS-11, MRZS-2, CRZS-3, CRZS-12, MRZS-10, ORTS-7, CRTS-7, CRTS-
+ 12, PRZS-10, PRZS-11, ORZS-8, ORZS-20, CRZS-2, PRTS-1,
ORTS-3, MRTS-1, MRZS-12
CRZS-5, PRZS-14, PRZ-38, ORZS-19, MRZS-13, MRTS-3, MRZS-3, MRZS-1, CRZS-
++ 10, CRZS-24, ORZS-17, PRZS-30
PRTS-5, CRZS-20, ORZS-3 ORZS-13, MRZS-18, PRZS-21
+++ ORZS-18, CRZS-23, MRZS-14
++++ CRZS-1, 6-9, 11, 13-19,22,25
- PRZS-1-9, 13, 15-20, 22-29,31-37
ORZS-1,2,4-7,9,10,12,14-16,20-27
MRZS-4-9,11,15-17,18,20
MRTS-2,4,5,6
PRTS-2,3,4
CRTS-1-6,8-11
ORTS-1,2,4-6,8-10

KEYS RHIZOSPHERIC SAMPLE; ORZS- OKRO

1. ORTS- OKRO ROOT SAMPLE; CRTS- CASSAVA RHIZOSPHERIC SAMPLE.
ROOT SAMPLE; PRTS- PLANTAIN ROOT SAMPE; 2. ZONE OF SOLUBILIZATION RANGING 0.0-1.5mm
MRTS- MAZIE ROOT SAMPLE; CRZS- CASSAVA = +; 1.5-3.0 = ++; 3.0-4.5mm = +++; >4.5mm = ++++;
RHIZOSPHERIC SAMPLE; PRZS- PLANTAIN
RHIZOSPHERIC SAMPLE; MRZS- MAZIE NO ZONE OF SOLUBILIZATION= -

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Volume 6, Issue 9, September – 2021 International Journal of Innovative Science and Research Technology
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3.2 Phosphate Solubilization Index (PSI) of Isolates isolate from okro rhizosphere sample (ORZS-11). The
The result of the phosphate solubilization index of isolate with the highest index was isolated from the cassava
positive isolates is presented in Table 2. The index is the rhizospheric sample (CRZS-23). About twenty percent (8)
ratio of the diameter of zone of clearance to the diameter of of the phosphate solubilizers isolated had PSI ≥ 140. These
colonial growth by a hundred. The result values range from isolates were subjected to further studies.
109.1 to 190. The isolate with the lowest index was an

TABLE 2: Phosphate Solubilization Index (PSI) of Isolates

Isolates Diameters of zone of Diameter of Colonial PSI (𝑫⁄𝒅 𝒙 𝟏𝟎𝟎)
Clearance Growth
PRZS-14 9.0 5.0 180.0
MRZS-14 8.5 5.5 154.5
CRZS-23 9.5 5.0 190.0
ORZS-11 6.0 5.5 109.1
CRZS-3 7.5 6.0 125.0
MRZS-10 9.5 7.0 135.7
CRZS-20 10.5 6.5 161.5
CRZS-5 7.0 5.5 127.3
MRZS-2 6.5 5.0 130.0
PRTS-5 7.5 5.0 150.0
ORZS-18 11.5 7.0 164.3
PRZS-21 9.0 5.0 180.0
MRZS-13 6.5 5.0 130.0
MRTS-3 7.5 5.5 136.4
ORZS-19 8.0 6.5 123.1
ORTS-7 8.5 6.5 130.8
MRZS-18 9.0 6.5 138.5
ORZS-3 9.0 6.5 138.5
ORZS-13 12.5 7.5 166.7
PRZS-38 10.0 8.0 125.0
MRZS-3 8.5 6.5 130.8
MRZS-1 7.0 5.5 127.3
CRZS-10 7.5 6.0 125.0
CRZS-24 7.5 5.5 136.4
ORZS-17 8.0 6.0 133.3
PRZS-30 8.5 7.0 121.4
CRTS-12 10.5 8.0 131.3
CRTS-7 9.0 6.5 138.5
PRZS-10 8.5 6.0 130.8
PRZS-11 7.5 5.0 136.4
ORZS-8 9.0 7.0 128.6
ORZS-20 8.5 6.5 130.8
PRTS-1 10.0 7.5 133.3
MRTS-1 7.5 6.0 125.0
ORTS-3 9.0 6.5 138.5
CRZS-2 8.5 7.0 121.5
MRZS-12 9.0 7.0 128.6

3.3 Identification of Bacterial Isolates with PSI≥140 grow in the presence of oxygen. Other biochemical tests
Using Colonial, Morphological and Biosynthetate carried out showed that Pseudomonas was positive for the
Characteristics following test: “citrate, gelatin hydrolysis, nitrate oxidase,
The morphological and biochemical characteristics of and test”. On the other hand, Pseudomonas was negative for
the bacterial isolates with PSI≥ 140 are presented in Table 3. indole, methyred urease, voges proskauer, maltose, glucose
The isolates with PSI≥ 140 were Pseudomonas sp. and sucrose. Pseudomonas does not produce gas and does
and Bacillus sp. Their morphological characteristics were not hydrolyze starch. The spore test revealed
recorded based on their size, shape, margin, elevation, that Pseudomonas is a non-spore former.
pigmentation, and color. Gram stain test revealed
that Pseudomonas sp is gram-negative as it retained the The result of the gram stain revealed that Bacillus sp
color of the counterstain used. Oxidase test was used to was a gram-positive organism because it retained the color
differentiate Pseudomonas from other gram-negative bacilli. of the primary stain (crystal violet) and was not decolorized
Catalase test was used to test the ability of Pseudomonas to by alcohol. It was found to be catalase, citrate, nitrate, voges

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proskauer , mannitol, maltose, glucose, sucrose and gelatin indole, methyl red, oxidase, and urease. The result of the
hydrolysis positive. Bacillus sp. showed a negative result for spore stain revealed that it is a spore former.

TABLE 3: Identification of Bacterial Isolates with PSI ≥ 140 using Colonial, Morphological and Biochemical
Characteristics
Isolates PRZS-14 MRZS- CRZS-23 CRZS-20 PRZS- PRZS-5 ORZS-18 ORZS-13
14 21
Colonial and
Morphological
characteristics

Size 2mm 3mm 3mm 3mm 3mm 2mm 3mm 3mm

Shape Rods Spherical Rods Rods Rods Rods Rods
Margin smooth edge Entire Irregular Irregular Entire smooth edge Irregular Irregular
Edge edge Convex edge edge
Elevation convex convex Flat Flat Convex Slightly Flat
Bluish green Convex
Colour bluish green Yellow Glassy Glassy Pale Glassy Glassy
Appearance appearance orange + appearance appearance
- - -
Pigmentation + - - - - + +
Gram reaction - + + +
Biochemical +
Test + + +
Catalase + + + + + + + +
Citrate + + + + + + - -
Gas + - - - - + +
Gelatin + + + + + -
hydrolysis - -
Indole - - - - + - -
Methyle red - - - - - - +
Nitrate + + + + + + +
reduction - -
Oxidase + - - + + - + +
Sporer - + + + + - + -
Urease - - - - - - +
Voges - + + + + + +
Proskauer - +
Mannitol + + + + + - +
Maltose - + + + + - + +
Glucose - + + + + + + +
Surcrose - + + + + - + +
Motality + + + + + Pseudomonas + +
Starch - + + + + sp. + Bacillus
Preliminary Pseudomonas Bacillus Bacillus sp. Bacillus sp. Bacillus + sp.
Identification sp. sp. sp. +
Bacillus
sp.

3.4 Identification of Fungal Isolates with PSI≥140 Using Cultural and Microscopic Characteristics
The result of the identification of fungal isolates with PSI≥ 140 is as presented in Table 4. The isolates were Aspergillus sp.
and Penicillium sp. Aspergillus sp. was found to be a powdery colony, with dark brown front colour. The reverse colour was also
brown. It had a flatty spread on the surface of the solid medium. Microscopically, Aspergillus sp. had septate and branched hyphae
with conidia that appeared in chains. Penicilliums sp. front colour was found to be grey with a large white border and white
reverse. Microscopically, Penicillium sp. has long branched septate conidiophores consisting of brown-like conidia in chains at
the tips of the phialides.

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Table 4: Identification of Fungal Isolates with PSI ≥140 Using cultural and Microscopic characteristics
Isolates Cultural Characteristics Microscopic Characteristics Preliminary Identification
MRTS-3 Powdery, dark brown, flatty Septate and branched hyphae Aspergillus sp.
spread and brown reverse. with conidia in chains.
MRZS-18
Grey colony with large white long conidiophores consisting Penicillium sp.
ORZS-3 border and white reverse. of brown like conidia in
Grey colony with large white chains. Penicillium sp.
border and white reverse. long conidiophores consisting
PRZS-38 of brown like conidia in Aspergillus sp.
Powdery, dark brown, flatty chains
spread on the surface of the
solid medium and brown
reverse. Septate and branched hyphae
with conidia in chains.

3.5 Phosphate Solubilization Efficiency of Isolates on the Green Revolution that save a billion people (Pingali,
Pikovskaya’s Agar 2012), is discovered to be inefficient. This is because 75-
The result of the Phosphate Solubilization Efficiency 90% of P fertilizer applied to soil is immobilized in
of Isolates on Pikovskaya’s Agar is presented in Fig 1. The precipitation reaction with Ca2+, Al3+ or Fe3+ depending
bar chart is the mean of triplicate solubilization indexes of on the soil pH (Goldstein, 1994). The high cost of the
the isolates in two days intervals. The bar chart reveals that fertilizer and its environmental degradation ability demands
bacterial isolates are more phosphate solubilizing efficiency the development of a sustainable biological-based source of
than fungal isolates. There were two bacterial genera P to avert the danger of permanently destroying the fertility
isolated: namely Pseudomonas, and Bacillus, with of soil through prolonged use of P fertilizer (Aliyat et al.,
efficiency order as follows: Pseudomonas> Bacillus. 2020). This study, which isolated and characterized
Among fungal isolates, Penicillium sp. is more phosphate Phosphate solubilizing microorganisms from the rhizosphere
solubilizing efficiency than Aspergillus sp. and the roots of crops indigenous to Ihiagw-Owerri , Imo
State Nigeria, was inclining to the new trend of sourcing P
biologically.

Whitelaw (2000) reported the importance of microbial

solubilizers in the maintenance of the global cycle. The
mechanisms used by these microbes include the excretion of
organic acids or the production of phosphatase enzymes
(Kucey, 1983). The medium used for the screening of the
isolates for phosphate solubilizing ability, Pikovskaya’s
agar, contains tricalcium phosphate (Ca3(PO4)2) an
insoluble phosphate. The ability of the isolates to solubilize
it produces a positive result which manifests as the zone of
clearance by 26.9% (39) of the 145 isolates screened. The
production of organic acids by these solubilizers was the
reason for the zone of clearance. The carboxylate of the
organic acids produced, through chelation and ligand
exchange, brought about the solubilization. The diameter of
clearance varied between 1.5-4.5mm among the isolates;
Fig. 1: Phosphate Solubilization Efficiency of Isolates on with 46.1% producing ≤ 1.5mm, 30.8%≤3.0mm, 15.4% ≤
Pikovskaya’s Agar 4.5, and 7.7% ≥ 4.5mm. The diameter could be proportional
to the concentration of organic acid produced.
IV. DISCUSSION
The phosphate solubilization index (PSI) of the
Phosphate plays an indispensable metabolic role in isolates ranged from 109.1 to 190. Isolates with PSI ≥ 140
plant biochemistry. It is of great importance in were 20% of the 39 isolates that could solubilize phosphate.
photosynthesis, respiration, energy storage, and transfer, cell Microbial identification protocol carried out on these
division and enlargement, and in several other processes in isolates revealed that 66.7% were bacterial while 33.3%
living plants. Below optimal P availability, plant yield is were fungal. The genera of microbes were Bacillus sp. were
impaired below economic level. The packaging of P and 75% of the bacterial isolates while Pseudomonas sp. was
other plant primary nutrients (nitrogen and potassium) into 25%. Fungal isolates were Aspergillus sp and Penicillium sp
chemical fertilizer, which was responsible for the success of with an equal frequency of occurrence.

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Bhattacharyya and Jha (2012) report of most cillus sp. can be optimized and developed into biofertilizer
significant bacterial solubilizers as Azotobacter, Bacillus (B. products for use as an alternative to chemical fertilizer. This
megaterium, B. circulans, B. subtilis, B. will ensure sustainable agro development in Nigeria and
polymyxa, Beijerinckia, Burkholderia, Enterobacter, open an agro channel for entrepreneurial microbiology.
Erwinia, Flavobacterium, Microbacterium, Pseudomonas
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