ISSN: 2084-3577
Journal of Biology
and Earth Sciences
BIOLOGY
ORIGINAL ARTICLE
Solubilization of inorganic phosphate by phosphate
solubilizing fungi isolated from Egyptian soils
Manal Mohamed Yasser*, Ahmad S. M. Mousa, Osama N. Massoud, Siada H. Nasr
Botany and Microbiology Department, Faculty of Science, Benisuef University, Egypt
ABSTRACT
Many fungal strains were recorded by using dilution plate methods on Pikovskaya's medium (PVK) from
soil samples were collected from different localities and different habitats in Beni-Suef governorate,
Egypt. Aspergillus, Fusarium, Penicillium and Trichoderma were the most important genera isolated
during this investigation. Phosphate solubilizing fungi (PSF) were selected by formation of halo zone
around fungal colony on (PVK) agar plate after 4 days of incubation. PSF were identified
morphologically and the most common genera were Aspergillus, Penicillium and Trichoderma. All
isolates exhibit different levels of phosphate solubilizing activity on (PVK) broth medium containing tricalcium phosphate (TCP) as sole phosphorus source. Final pH of the medium, solubilized P and dry
weights of fungi were recorded. Aspergillus niger, Penicillium variable and Trichoderma harzianum 301
showed solubilization potential or activity 1 .67, 0.55% and 0.32% respectively whereas the pH values of
the culture filtrates were 3.6, 4.3 and 5.0, respectively.
Key words: Phosphate; Fungi; Aspergillus spp.; Penicillium spp. ; Trichoderma spp.
J Biol Earth Sci 201 4; 4(1 ): B83-B90
* Corresponding author:
Manal Mohamed Yasser
Email: [email protected]
Phone: 002 01 006277643, 002 0822 354556
Fax: 002 0822 334551
Original Submission: 1 8 February 201 4; Revised Submission: 1 7 March 201 4; Accepted: 1 9 March 201 4
Copyright © 201 4 Author(s). Journal of Biology and Earth Sciences © 201 4 T.M.Karpiński. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
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Yasser et al. Solubilization of inorganic phosphate by fungi isolated from Egyptian soils
INTRODUCTION
MATERIALS AND METHODS
Phosphorus (P) is one of the most important
macro elements after nitrogen for both plant and
microorganisms. It is structural and functional
element as well as energy transfer. In general,
Egyptian soils have low phosphorus appearance
that cannot cover the demands of plant [1 ]. The
application of chemical fertilizers to the soil is not
successful pathway because it is rapidly fixed
where it react with iron, aluminum and manganese
in acid soil and with calcium in neutral and alkaline
soil and precipitated strongly to the surface of the
soil particles, this reaction remove available phosphorus from soil solution and become unavailable
to plant [2].
In order to overcome this obstacle by safe ways,
less expensive costs and friendly environment,
there are groups of soil microorganisms including
bacteria, fungi, actinomycetes and arbuscular
mycorrhiza have the ability to solubilize the precipitated phosphates, converting them into soluble
forms H 2PO 4- and HPO 42- that are available to
plant [3]. Microbial solubilization of insoluble phosphate occurs by different mechanisms, such as
acidification, chelation, ion-exchange reactions and
polymeric substances formation [4]. There are two
ways in microbial P solubilization: by solubilization
processes [5], and from P accumulation in the
biomass of microorganism [6]. The important genera of (PSF) are Aspergillus [7-1 0] and some species of Penicillium [11 -1 4]. PSF do not lose the P
dissolving activity upon repeated sub culturing
under laboratory conditions [1 5]. Penicillium oxali­
cum was isolated from rhizosphere of rock phosphate mine and tested for its ability to solubilize
rock phosphate and promote the growth of wheat
and maize. The solubilization was increased with
the increase in concentration of rock phosphate
[1 6]. The PSF can be isolated from rhizospheric,
non rhizospheric soil, phyllosphere, rock phosphate
deposit area soil and polluted soil [1 7]. In the
present study, we isolated the most efficient phosphate solubilizing fungi from various soils to assess their phosphorus solubilizing capacity from
inorganic phosphate sources and we can use
the organisms with higher phosphate solubilizing
abilities as economically sound alternative way
instead of the more expensive superphosphates
and thus posses a greater agronomic utility.
Collection of soil samples
Soil samples were collected from different localities in Beni-suef governorate, Egypt. Each soil
sample was taken from the profile of (0-1 0 cm in
depth) by using sterile auger. The soil samples were
transferred to laboratory in sterile polyethylene bags
under aseptic conditions. The soil samples were reserved in refrigerator at 5ºC.The soils dried at room
temperature and their physico chemical properties
were determined. Soil pH and E.C. were determined in 1 :2 soil-water suspensions.
Screening of phosphate solubilizing fungi
Phosphate solubilizing fungi were isolated by
the dilution plate methods modified by Johnson et
al. [1 8] on PVK medium [1 9] with tri-calcium phosphate as insoluble inorganic phosphate source.
Rose Bengal as bacteriostatic was added (1 0 ml/l)
at concentration 1 /1 5000 [20].
Total fungal counts were calculated in triplicate
after 7 days of isolation by multiplying average
number of colonies in each plate with inverted
dilution factors. The isolates were identified on
the basis of colony morphology, spore characteristics and microscopic examination according to
Moubasher [21 ].
Selection of phosphate solubilizing fungi
PVK medium without rose Bengal addition was
prepared. Sterilized PVK media was poured into
sterilized plates, after solidification of the media, a
pinpoint inoculation of fungal strains was placed on
the center of plates under aseptic conditions. They
were incubated at 28 ± 2ºC for 4 days with continuous observation for colony diameter. The P solubilizing fungi were detected by the formation of clear
halo around their colonies (Fig. 1 ). Solubiliztion index (SI) was calculated according to the ratio of
total diameter (colony and halo zone) and colony
diameter [22].
Quantitative estimation of phosphate solubilization
Many isolates which did not show clear zone on
agar plates, solubilized phosphates in liquid medium [23-24] as in case of Trichoderma. The solubiliztion activity of Trichoderma was measured in PVK
broth medium as shown in Table 4. Solubilization
activity was carried out in 1 00 ml PVK broth me-
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Yasser et al. Solubilization of inorganic phosphate by fungi isolated from Egyptian soils
dium amended with 0.5% tri-calcium phosphate.
1 ml spore suspension of each fungal culture
(1 0 9 CFU/flask) of four days old on Czapek's agar
medium added to each flasks containing broth
medium in triplicate. Control was prepared without
fungal inoculation. Flasks were incubated in shaking
incubator (1 20 rpm/min) for 7 days at 28°C. Cultures were harvest to record changes in pH and dry
weights of fungi.
Determination of solubilized P
P in solution was extracted with ammonium bicarbonate diethylene triamine penta acetic acid
method [25] and the available phosphorus in culture
filtrate medium was estimated by Watanabe and
Olsen [26]. The extract of available phosphorus was
calculated from standard curve prepared using various concentration of standard KH 2PO 4 solution the
results were expressed as µg ml -1 .
RESULTS AND DISCUSSION
Screening of phosphate solubilizing fungi
A summary of the fungi isolated during this
investigation, their total counts and their number
of cases of isolation of each as well as their
degree of occurrence are presented in Table 1 .
The most common genera isolated were Asper­
gillus, Fusarium, Penicillium and Trichoderma according to the degree of occurrence. The 1 73 fungi
taxa isolated from thirty soil samples, only ten
isolates showed significant zone of phosphate
solubilization on PVK agar medium as shown in
Table 3. Clear zones were formed around the
colonies after 5 to 7 days of incubation on solidified PVK medium supplemented with TCP, indicating phosphate-solubilizing ability of the fungal
isolates Fig. 1 (criteria were used to select isolates).
The results illustrated in Table 2 showed that
only eleven samples from thirty soil samples
collected from different localities in Beni-Suef
governorate possessed PSF. They were isolated
from canal bank, cultivated, reclaimed soil and
orchard as illustrated in Table 1 . The isolation of
PSF from saline soil refers to the ability of these
isolates to high and salinity tolerance, these results
were confirmed by Rajankar et al. [27] who isolated PSF from saline, and he suggested its application as bio-fertilizer should be helpful to decrease
the salinity of soil by neutralization process, because these microorganisms release acid in very
minute quantity during phosphate solubilization.
Fig. 1. Clearing zones around colonies of (A) Aspergillus niger 2, (B) Aspergillus japonicus
(D) Aspergillus carbonaceous, (E) Aspergillus niger 1 , (F) Penicillium variable.
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4, (C) Aspergillus japonicus 6,
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Yasser et al. Solubilization of inorganic phosphate by fungi isolated from Egyptian soils
Total count, the number of cases of isolation and occurrence remark of fungal genera
and species recovered from 30 soil samples on PVK medium (colonies per mg of dry soil).
Table 1.
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Yasser et al. Solubilization of inorganic phosphate by fungi isolated from Egyptian soils
Table 1.
continued.
Legends: H = high occurrence remark (1 5-30 No of cases), M = moderate occurrence remark (8-1 4 No
of cases), L = low occurrence remark (4-7 No of cases), R = rare occurrence remark (1 -3 No of cases).
Table 2.
Egypt.
Characteristics of soil samples which contain phosphate solubilizing fungi (PSF) in Beni-Suef governorate,
Solubilization index of PSF on solid agar plate
Table 3 showed the solubilization Index (SI) of
the tested phosphate solubilizing fungal strains ranged between 1 .05 to 1 .45 in the present study after
4 days of inoculation. Clear zone around the fungal
colony increase in diameter by putting the Petri-dishes inoculated with fungi in refrigerator at 5°C and
also increase by the time, this is due to fungi produce cold active metabolites at low temperature
[28]. Results showed that clear zone reached 5mm
among A. niger and A. japonicus while in case of
P. variable, P. funiculosum and P. purpurogenum
obtained clear zone reached 2 mm. Colony diameters of Aspergillus species were greater than colony
diameters of Penicillium species as a result SI
among Penicillium spp. was greater than SI among
Aspergillus spp. as shown in Table 3. These findings in the contrary to the result of El-AzouniI [29],
who reported that SI of A. niger was greater than
P. ilalicum. Sometimes abrupt changes or no constant pattern for P-solubilization occurred in these
values. The zone of clearance was due to solubilization of tri-calcium phosphate which is the result of
combined effect of pH decrease and organic acids
production [30]. Microorganism that produce zone
of clearance around its colonies in plate assay
method selected as a phosphate solubilizer.
Liquid culture experiments (Quantitative estimation of phosphate solubilization) showed that all
isolates of Aspergillus spp. and Penicillium spp.
caused a remarkable drop in pH of culture media
and they solubilized considerable amounts of phosphates compared to the control (not inoculated
with fungi), where control remained constant around
7.0 and available P was estimated to be about
0.36%. In present investigation, A. niger 2 and
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Yasser et al. Solubilization of inorganic phosphate by fungi isolated from Egyptian soils
Solubilization activity of some identified fungal isolates isolated from
different soil samples.
Table 3.
Legends:
(+) = 2 mm
(++) = 2-4 mm
(+++) = 4-5 mm
Changes in PH and biomass dry weight (g) during tri-calcium
phosphates (TCP) (%) solubilization for different Trichoderma spp.
Table 4.
* Each value is the
means of three replicates
± standard error
P. variable
were showed phosphate solubilizing
activity followed by T. harzianum and T. koningii.
The pH drop in P-solubilizing fungi liquid cultures
resulted in present study was supported by ElKatatny [31 ]. Also Pradhan and Sukla [32] stated
that Aspergillus spp. showed much higher drop in
pH and high P solubilization when compared to
other genera and most of the previous reports state
that calcium phosphates are dissolved by acidification. Therefore, any microorganism that acidifies its
external medium will show some level of phosphorus solubilizing activity. It was found that more amount phosphorus was released 1 .67% when the pH
of the culture filtrate was acidic 3.67 but others have
contradictory reports that solubilization is not always
proportional to the decline in pH [33]. The available
P was 0.87% at pH 4.4 in case of A. japonicus 3,
while the available P was 0.37% at pH 4 in case of
P. purpurogenum. The differences in drop in pH by
the two strains indicate the varying diffusion rates of
different organic acids secreted by these two tested
strains of fungi.
The overall results of the study indicate that acid
production was not the only reason for phosphate
release into the medium that might be acidification
either by protone extrusion associated with ammonium assimilation [34] or by organic acids production and proton extrusion [35]. Acid phosphatases
and phytases secreated by these microorganisms
also have an important role in phosphate solubilization [36].
Phosphate solubilization by
res
Trichoderma
cultu-
Disappearance of tri-calcium phosphate (TCP)
in the culture filterate after 7 days indicated the
high activity of Trichoderma for solubilization of
inorganic bound phosphate (TCP), which might
have been subsequently taken up by the fungus for
cellular processes, Table 4. Decrease in phosphate
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Yasser et al. Solubilization of inorganic phosphate by fungi isolated from Egyptian soils
concentration might be correlated with its sequestration by Trichoderma mycelium, to be released
in a readily available from in close proximity to
the roots after lysis of mycelium with age [37].
T. harzianum 301 and T. koningii with low dry
weight 0.23 and 0.45 g respectively solubilize more
P 0.32% while T. longibrachiatum and T. pseudo­
koningi solubilize less P 0.23 and 0.21 % respectively, whereas they showed dry weights 0.80 and
0.45 g respectively. The pH value never reached
below 4.8 in comparison to Aspergillus and Peni­
cillium which reached 3.6 in case of A. niger 2
and reached 4 in case of P. purpurogenum. Slightly
decrease in pH is conformity with Ejikeme et al. [38]
who studied the efficiency of solubilization of tricalcium phosphate (TCP) by phosphate solubilizing
fungi (PSF) isolated from Nsukka pepper plant
rhizosphere and root free soil. These pH reductions
are due to secretion of organic acids excreted by
PSF [39].
CONCLUSIONS
Results obtained with the present study shows
that Aspergillus niger 2 and Penicillium variable are
the most efficient fungal strains as far as their
phosphate solubilizing activity further study are
being done with these fungal isolates to observe
their potential to improve the productivity of plants
under the field conditions.
TRANSPARENCY DECLARATION
The authors declare no conflicts of interest.
AUTHORS' CONTRIBUTION
MMY: Conception and design; MMY and ASM:
Development of methodology; MMY: Acquisition
of data; MMY and SHN: Analysis and interpretation
of data; MMY, ASM and ONM: Writing, review
and/or revision of the manuscript; Administrative,
technical, or material support; ASM: Study supervision. All Authors are involved in drafting the
manuscript, read and approved the final version
of the manuscript.
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