Isolation and Charecterisation of Seaweed Endophytic Fungi as an Efficient Phosphate Solubiizers

Abstract

Phosphate-solubilizing fungi (PSF) generally enhance the availablility of phosphorus (P) released from soil, which contributes to plants' P requirement, especially in P-limiting regions. In this study we isolated endophytic fungi from seaweeds and screened for phosphate solubilizng in both solid and liquid culture and estimated the solubilizing index and enzyme activity. Six fungus of Penicillium oxalicum, P.citrinums and Aspergillus sp. shows maximum phosphate solubilizing activity. Hence Seaweed endophytic fungus isolated from chlorophyceae express as an alternate source to replace chemical fertilizer.

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BIOSCIENCES BIOTECHNOLOGY RESEARCH ASIA, March 2019. Vol. 16(1), p. 33-39
Published by Oriental Scientific Publishing Company © 2018
This is an Open Access article licensed under a Creative Commons license: Attribution 4.0 International (CC-BY).
*Corresponding author E-mail: noorbiotek@gmail.com
Isolation and Charecterisation of Seaweed Endophytic
Fungi as an Efficient Phosphate Solubiizers
A. Noorjahan, B. Aiyamperumal and P. Anantharaman.
CAS in Marine Biology, Faculty of Marine Sciences,
Annamalai University, Parangipettai-608502, India.
http://dx.doi.org/10.13005/bbra/2718
(Received: 07 March 2019; accepted: 28 March 2019)
Phosphate-solubilizing fungi (PSF) generally enhance the availablility of phosphorus
(P) released from soil, which contributes to plants’ P requirement, especially in P-limiting
regions. In this study we isolated endophytic fungi from seaweeds and screened for phosphate
solubilizng in both solid and liquid culture and estimated the solubilizing index and enzyme
activity. Six fungus of Penicillium oxalicum, P.citrinums and Aspergillus sp. shows maximum
phosphate solubilizing activity. Hence Seaweed endophytic fungus isolated from chlorophyceae
express as an alternate source to replace chemical fertilizer.
Keywords: Chlorophyceae; Penicillium Oxalicum; Aspergillus sp.
Phosphorus (P) is the next important
nutrients after nitrogen which have an impact on
plant growth and its metabolic processes (Widawati
S & Suliasih D, 2006). Phosphorus is essential
to promote photosynthesis, energy regulation
,sugar production, synthesis of nucleic acid, and
enhancing N2 xation in leguminous plants (Saber
et al.,2005) . It also plays an important role in
strengthening straw of the cereal plant and quality
of the crop, boost ower formation and increase
the fruit production, stimulates root development
and essential for seed formation, maturity, and also
to develop resistance against diseases (Sharma et
al., 2011, Richardson, 2007).
Generally, the mobility of phosphate
ions in the soil is very low due to high retention.
Stevenson and Holford reported in 1986 that the
plants utilize P from soil in the rate of about 10-30%
(Stevenson,1986) and the remaining 70-90% of
Phosphate is accumulated in the soil as immobilize
form or it is bounded with Al or Fe in acid soils or
Ca and Mg in alkaline soils (Prochnow et al., 2006;
Yang et al.,2010)
This insoluble form of Phosphorus is
converted to soluble form by the microorganisms
which play a key role in dissolving both free and
bound phosphate in the soil that is environmentally
friendly and sustainable (Khan et al.,2007).
Microorganisms including fungi, bacteria and
actinomycetes are known to be efcient as xed P
solubilizes (Sundara et al., 2002).
Seaweeds are considered as a well-
endowed determinant for nutrients that ultimately
leads to high competition between different
microbial communities (Burgess et al.1999;
Armstrong et al. 2001; Penesyan et al. 2009).

34 NOORJAHAH et al., Biosci., Biotech. Res. Asia, Vol. 16(1), 33-39 (2019)
The second largest diverse assemblage of marine
fungi is from seaweeds (Bugni and Ireland 2004;
Schulz et al.2008; Suryanarayanan et al. 2010;
Godinho et al. 2013). Seaweeds encompassing
genera belonging to Phaeophyceae, Rhodophyceae
and Chlorophyceae have been broadly studied
worldwide for their fungal associations and the
study revealed that the dominant species of
fungal endosymbionts were ascomycetes and
anarmorphic fungi (Schulz et al. 2008; Zuccaro
et al. 2008; Suryanarayanan 2012; Godinho et
al. 2013;). The fungi associated with seaweeds
are mostly parasites, saprobes or asymptomatic
fungal endosymbionts (Bugni and Ireland 2004;
Loque et al.2010; Suryanarayanan et al. 2010).
The diverse fungal population in Red and Brown
seaweeds were comparatively high compared to
that of Green seaweed (Suryanarayanan et al.
2010). This is due to short lifecycle of Green
seaweeds and characteristically slow growth of
the endosymbionts could together be responsible
for low fungal diversity (Zuccaro and Mitchell
2005). Fungal genera of Acremonium, Alternaria,
Arthrinium, Aspergillus, Cladosporium, Fusarium,
Geomyces, Penicillium, and Phoma (Zuccaro et
al. 2003; Loque et al. 2010; Suryanarayanan et al.
2010; Flewelling et al. 2013; Godinho et al. 2013;
Furbino et al.2014) were the most common fungal
endosymbionts.
On comparing with bacteria fungi
have greater ability to solubilize insouble
phosphate(Nahas, 1996). Endophytic fungi from
Marine plants and Marine algae are gaining interest
because of their existence in an ecosystem renowed
by resource inconstancy such as temperature,
salinity, osmotic stress, light vailabity (Debbab et
al., 2011; Oliveira et al., 2012).
Therefore, the objective of this study is
to search for the seaweed in order to select the
endophytic fungi which can solubilze phosphate.
The results may provide insights into potential
fertilizer in acidic soils and P-deficit soils.
Microbial phosphate solubilization has an
impact on plant growth promotion. There are
several reports regarding plant growth promotion
due to inoculation of phosphate solubilizing
microorganisms. Screening and characterization
of phosphate solubilizing microorganisms are
important for proper utilization of their benecial
effects to increase the crop production and sustain
agricultural productivity of the country without
contaminating environments.
MATERIALS AND METHODS
Collection of sample
Seaweeds were collected from
Rameswarem coastal region. Abundant seaweeds
are selected for the isolation of endophytic fungus.
Cauerapa racemosa, Halimeda macrooba
(Chlorophyceae), Turbinaria conoides, Sargassum
sp, Padina sp (Phaeophyceae), Gracilaria
sp., Portieria sp. (Rhodophyceae). Seaweeds
were collected in sterile bags and processed in
laboratory. Freshly collected plant materials were
utilized for the isolation of the endophytic fungi to
reduce the chance of contamination.
Isolation of endophytic fungi from seaweeds
Healthy thallus of the seaweeds were
thoroughly washed in seawater followed by
running tap water, then surface sterilized by a
modied method of [Raviraja, 2005]. The selected
thallus segments were immersed in 95% ethanol
for 30 sec, 4% sodium hypochlorite solution for 3
min and 95% ethanol for 30 sec followed by rinsing
with sterile distilled water three times and allowed
to surface dry under sterile conditions. After drying,
each thallus segment was cut into approximately
0.5 cm and placed on petri plates containing potato
dextrose agar medium (PDA) supplemented with
Chloramphenicol (100 mg/L) to suppress the
bacterial growth. Petri plates were sealed with
paralm and incubated at 30°C in a light chamber
for up to one week. They were monitored every day
for growth of endophytic fungal colonies. Fungi
growing out from the samples were subsequently
transferred onto fresh PDA plates to isolate pure
colonies.
Identication by morphological characteristics
of endophytic fungi
Sporulating fungi were identied based on
colony morphology, conidiospore and conidiophore
characteristics. The microscopic identication of
the isolates was carried out by lacto phenol staining
technique [Nagamani et al., 2006].
Screening of Phosphate solubilizing in Solid
Medium
Phosphate solubilizing ability of the
isolates was conrmed by incubating them on
Pikovskaya’s agar medium incorporated with

35 NOORJAHAH et al., Biosci., Biotech. Res. Asia, Vol. 16(1), 33-39 (2019)
S. Fungal Isolates Phosphate Solubilizng Index (PSI) mm
No 7th Day 14thday 21st day
1 Pencillium oxallicum 3.5 5.2 7.1
2 P. Purpurogenum 2.5 3 3.5
3 P.citrinum 2.6 3.2 3.7
4 P.aurantio-grieseum 1.3 2.4 2.9
5 Aspergillus niger. 1.7 2.6 3.1
6 Aspergillus sp. 3.2 5.7 7.9
Fig. 1. Solubilizing index
5% tri-calcium phosphate at 28 ± 2°C for 15
days. Diameter of clearance zone was measured
successively after 24 h, up to 15 days. The
Solubilization Index (SI) is the ratio of total
diameter i.e. clearance zone including fungal growth
and the colony diameter. All the observations were
recorded in triplicate. Pikovskaya, 1948.
P-solubilizing capacity under different pH
conditions
After preparation of the conidial
suspensions of the fungal strains, was inoculated
into 50-mL asks containing 30 mL of Pikovskaya’s
liquid media, were added to a concentration of 5
g/L, respectively. The pH of the Pikovskaya’s
liquid media was adjusted to 3.5, 4.5, 5.5, and
6.5 with 2 mol/L hydrochloric acid, respectively,
before addition of the P sources the medium was
sterilized at 121°C for 20 min. Then, 0.5 mL of
the test fungal spore suspension was inoculated
into 50 mL triangular asks at various pH and
incubated at 25°C and 120 rpm for 8 d on a shaker.
The control comprised ask with uninoculated
medium. All treatments were centrifuged at 10,000
×g for 10 min, and the supernatant was used for
the measurements of soluble P and pH. The content
of soluble P in the supernatant was determined by
the colorimetric molybdate blue method (Olsen &
Sommers, 1982). All experiments were performed
in triplicate.
Estimation of Phosphatase activity
Phosphtates were estimated at weekly
intervals about 30 days. The test fungal isolates
were fermented in Potato Dextrose broth at 21°C
for 4 weeks. The fungal broth was ltered through
Whatman no. 42 lter paper and homogenized in a
pestle and mortar at 4 °C using 0.02 M Tris buffer
(1:1 w/v, pH 7.5). The macerate was centrifuged
at 16,000g for 20 min and the supernatant was
collected and estimated for Alkaline and Acid
phosphates.
For assaying alkaline phosphatase, to 0.1
mL the enzyme extract (the supernatant) 0.5 mL
of tris citrate buffer (pH 8.5/5.5 mM) was added
followed by 0.1 mL of MgCl2 (0.1 M) and then
1.0 mL of p-Nitrophenol phosphate (1 mg/mL).
The mixture was incubated for 30 min at 21°C and
Read at 405 nm. Same procedure was followed for
estimating acid phosphatase assay but an acetate
buffer (pH 4.5/0.1 M) was used in place of tris
citrate buffer. The tubes were incubated at 21°C
for 30 min. After incubation, 5 mL of 0.5 M NaOH
was added and the release of p-Nitrophenol was
measured at 405 nm. The values were converted to
micromoles of p-Nitrophenol with reference to the
standard curve. One enzyme unit was dened as the
amount of enzyme that catalysed the formation of 1
lmol of end product (p-Nitrophenol) in 1 min under
experimental conditions (Tabatabai and Bremner
1969).

36 NOORJAHAH et al., Biosci., Biotech. Res. Asia, Vol. 16(1), 33-39 (2019)
Fig. 2. Phosphate soubilization in pH of broth at 21°C estimated upto day 30 days at Weekly intervals
RESULTS AND DISCUSSIONS
Seven seaweeds belonging to
Chlrophyceae, Rhodophyceae and Phaeophyceae
were selected for the isolation of endophytic fungi.
Totally18 endophytic fungi were isolated and
screened for Phosphate solubilization. Of that six
species shows better result which is discussed in
this paper.
Phosphate solubilization Solid Medium
Endophytic fungal isolates showed
phosphate Solubilizing activities as detected in
Pikovskaya’s agar amended with tri-calcium
phosphate in the medium by the appearance of halos
around the inoculum on the medium. Aspergillus
sp, P. oxalicum, P. citrinum, superior solubilization
index (PSI) 5.7,5.2 and 3.2 respectively in 15 days
incubation.
Phosphatase solubilization in liquid culture
Phosphate solubilization by six species of
fungal strains was carried out at, The fungal broth
were weekly withdrawn to estimate solubilization
index and the corresponding changes in the pH of
the broth are presented in Fig. 3.
Phosphate solubilization was estimated
on the 7th day giving the values of Penicilium
oxallicum 220 µg mL–1 with the pH 3.9 ,by pH
3.3 it solubilize 110 µg mL–1 P. aurantio-griseum,
230 µg mL–1 by P.citrinum with an pH 3.7, 210 µg
mL–1 by P. purpurogenum pH 3.7 and Aspergilus
niger with the pH 4 it solubilize phosphate 190
µg mL–1 and Aspergillus sp. by 195 µg mL–1 with
the pH 3.5.
There is a increase in solubilization rate
and decrease in the pH at the 15th day of incubation.
The values were recorded as pH 3.3 with 310 µg

37 NOORJAHAH et al., Biosci., Biotech. Res. Asia, Vol. 16(1), 33-39 (2019)
Fig. 3. Acid and Alkaine phosphatase activity of Fungal species estimated weekly intervals upto 4 weeks
mL–1 P. oxalicum and 425 µg mL–1 with the pH
3.1 P. citrinum and at pH 3.1 P. purpurogenum
solubilizes 391 µg mL–1 for Aspergillus niger at 3.9
pH it solubilizes 410 µg mL–1 and Aspergillus sp.
with pH 3.5 solubilizes 325 µg mL–1 If the strains
attained the maximum solubilization rate at 400
µg mL–1 decline values were recorded at 21st day.
P.oxalicum attain its maximum solubilization at
30th day of incubation.
Decrease in pH of the broth coinciding
with increase in phosphate solubilization
estimations which indicates the production of
organic acids in the broth, recorded in all the
strains were noticed . Various mechanisms have
been reported for phosphate solubilization, the
most recognized one is through the production of
organic acids (Nahas,1996). Production of organic
acids, like citric, gluconic and oxalic acid, have
been recognized for phosphate solubilization by
several species of Penicillium, namely, P. bilaii,
P. radicum, P. rugulosum, P. variabile (Asea et
al., 1988; Cunningham and Kuiack 1992; Vassilev
et al. 1996). Omar (1998) reported phosphate
solubilization activity by Aspergillus niger and
Penicillium citrinum causing remarkable drop in
pH of liquid culture.
Quantitative estimation enzyme phospatase
The acidic and alkaline phosphatase
activity exhibited by six fungal stains were
incubated at 21°C for 30 days and estimated at
weekly interval is presented in Fig. 4. All the
species showed acidic phosphatase activity higher
(1.5–2.0 times) in comparison to the alkaline
phosphatase. The maximum phosphatase activity

38
NOORJAHAH et al., Biosci., Biotech. Res. Asia, Vol. 16(1), 33-39 (2019)
21.23 (Aspergillus sp.), 23.37(P. oxalicum) and
19.13 (Aspergillus niger), 16.32 (P. aurantio-
griseum) was recorded at the week. After week
2 and 28.83 (P. citrinum), 24.32 (Aspergillus sp.),
24.67 (P. oxallicum), 21.34 (P. purpurogenum) and
22.41 (Aspergillus niger) after week 3 of growth.
Alkaline phosphatase activity ranged between 9.67
(Aspergilus sp.) and 11.21 (P. citrinum) after week
3 of incubation. Tarafdar et al. (2003), reported the
efciency of seven fungal phosphatase producing
fungi and in which the acid phosphatase was
three times higher than the alkaline phosphatase.
Naturally occurring phosphate solubilizing
microorganisms have been recognized as a
source of P fertilizer. Amongst fungi, maximum
Penicillium species have been reported for various
properties of biotechnological applications.
CONCLUSION
Therefore, these strains can be a good
candidate and exploited as bio fertilizers through
further evaluation and optimization test to
increase agricultural productivity. Other strains
such as Aspergillus niger, P. aurantio-griseum,
P. purpurogenum were positive for phosphate
solubilization efficiency. P. oxalicum was the
superior among the isolated fungi in solubilizing
index 5.3 after further evaluation on invitro test,
green house and eld trials as bio fertilizer. The
rise in the cost of chemical fertilizer, the lack of
fertilizer industries in developing countries and
the growing environmental issue and biodiversity
loss using chemical fertilizer timely important
concern using alternative ecofriendly bio fertilizer
to increase yield and productivity
Fungus from marine ecosystems are
distinct from those of terrestrial environments.
The most efcient phosphate solubilizing strains
penicillium sp and Aspergillus sp. were identied
from green seaweed and to solubilise inorganic
phosphate to organic form. Phosphate solubilizing
fungus can effectively replace chemical fertilizer.
ACKNOWLEDGEMENT
We greatly thankful to DBT-BIRAC for
funding the project entitiled “Development of
integrated product with plant growth and defence
potential through end to end utilization of marine
biological resources”. Ref BT/SBIRI1394/31/16.
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