pISSN 2302-1616, eISSN 2580-2909

Vol 9, No. 1, June 2021, pp. 93-101

Available online http://journal.uin-alauddin.ac.id/index.php/biogenesis
DOI https://doi.org/10.24252/bio.v9i1.21216

Identification of endophytic fungi in nutgrass (Cyperus rotundus L.)

as solubilizing phosphate and indole-3-acetic acid producers
Nur Kusmiyati1*, Septian Tri Wicaksono1, Durrotul Maknuna1
1
Department of Biology, Faculty of Science and Technology, Universitas Islam Negeri Maulana Malik Ibrahim
Jl. Gajayana No. 50 Lowokwaru, Malang, East Java, Indonesia. 65144
*Email: nurkusmiyati_bio@uin-malang.ac.id

ABSTRACT. Low phosphate content in the soil causes insufficient plant needs. Meanwhile, endophytic
fungi in nutgrass have great potential as a phosphate solvent and produce indole-3-acetic acid (IAA).
Therefore, this study aims to determine the solubilizing phosphate and IAA production by the endophytic
fungi of nutgrass and identify the isolates based on rDNA-ITS sequences. Endophytic fungi isolates were
cultured in 10 ml of Pikovskaya broth media with Ca5(PO4)3 as the inorganic phosphate source. The PCR
results were analyzed using 1.5% agarose gel electrophoresis followed by sequence analysis. The isolation
and purification results showed five isolates coded URT1, URT2, URT3, URT4, and URT5, while the
solubilizing phosphate levels ranged from 54.03-87.83 ppm, with the highest levels found in the URT4
isolate. Furthermore, the IAA levels ranged from 5.58-45.50 ppm, with the highest levels produced by the
URT1 isolate. The molecular analysis with rDNA-ITS sequences showed that URT4 had 97.42% similarity
to Aspergillus terreus species, while UTR1 had 100% similarity to Fusarium oxysporum species. Based on
the results, the endophytic fungi of nutgrass from A. terreus and F. oxysporum species have great potential
as biofertilizers due to the high solubilizing phosphate and IAA levels.

Keywords: Aspergillus terreus; biofertilizers; Fusarium oxysporum; phylogenetic tree analysis; rDNA-ITS
sequences
Article History: Received 16 February 2021; Received in revised form 30 April 2021; Accepted 30 May 2021; Available online 30
June 2021
How to Cite This Article: Kusmiyati N, Wicaksono ST, Maknuna D. 2021. Identification of endophytic fungi in nutgrass (Cyperus
rotundus L.) as solubilizing phosphate and indole-3-acetic acid producers. Biogenesis: Jurnal Ilmiah Biologi. vol 9(1): 93–101. doi:
https://doi.org/10.24252/bio.v9i1.21216.

INTRODUCTION negative effects. Besides, the use of long-term

Most tropical countries usually have a very chemical phosphate fertilizers above normal
large swamp area divided into low and tidal dosage has negative impacts on the
swamps. Meanwhile, the expansion of environment, such as a decrease in soil quality,
agricultural areas to lands with acid sulfate soils soil fertility, and crop yield (Jiao et al., 2012;
is not an option, but a demand for the future. Kaur & Reddy, 2015; Ning et al., 2017).
However, the common problem with acid George et al. (2019) and Simarmata et al.
sulfate soils is the low availability of phosphate (2020), stated that endophytic microorganisms
nutrients. Phosphate constitutes one of the have great potential to promote growth and do
macromolecular nutrients needed in the soil to not cause any pathogenic reaction to the host
meet plant needs. Furthermore, it is very plant. Furthermore, phosphate-solubilizing
important, especially for root growth microorganisms synthesize phosphates which
stimulation, starch metabolism, assimilation are chemically and biologically undissolved.
process, elevation of homogeneous Zega et al. (2018) reported that 15 endophytic
microspores, viable pollen and ovules, bacteria of nutgrass (Cyperus rotundus L.)
acceleration of flowering, and ripening of seeds dissolved phosphate and produced indole-3-
or fruits (Ponnu et al., 2011; Kasno & Sutriadi, acetic acid (IAA), but the levels were only
2012; Ghanem et al., 2014). The phosphate moderately high. Nutgrass is a commonly
requirement is inversely proportional in the known agricultural weed in the world due to its
soil, meanwhile, the availability of dissolved fast reproduction, high survival ability, wide
phosphate is usually very low therefore, it is not distribution, and difficult growth control (Dor
sufficient to meet plant needs. The application & Hershenhorn, 2013; Coleman et al., 2015;
of phosphate fertilizer is a common alternative Peerzada, 2017). However, it contains several
often used to overcome this problem but it has important compounds, such as polyphenols,

Copyright © 2021. The authors. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
Kusmiyati et al. Biogenesis: Jurnal Ilmiah Biologi 94

flavonoids, tannins, and sterols which are g, MnSO4 0.0002 g dissolved in 1 L sterile
useful for antimicrobials, fungicides, water. Furthermore, the cultured isolates were
bactericides, antigenotoxic, and antioxidants incubated in a shaker at 25°C and a speed of 160
due to the symbiotic endophytic fungi present rpm for 7 days. The isolates were then filtered
in the plant (Kilani-Jaziri et al., 2011; Seo et al., using sterile Whatman filter paper and the
2011; Peerzada et al., 2015; Masfria & Permata, filtrate was centrifuged at 10000 rpm for 15
2018; Horn & Vediyappan, 2021). min, while 0.4 ml of the supernatant was taken
Endophytic fungi are microorganisms with and placed into a test tube. Molybdate
higher phosphate solubility compared to other phosphate color reagent was added 0.16 ml of
microorganisms (Klaic et al., 2017; Mehta et 2.5% sodium molybdate, 0.04 ml of 10 N
al., 2019). George et al. (2019) reported that sulfuric acids, 0.1 ml of 0.5 M hydrazine
rhizome endophytic fungi produce useful active sulfate, and 2.3 ml of sterile distilled water, and
compounds for plant growth and development, left to stand for 10-35 min. A positive result is
such as dissolving phosphate and releasing IAA indicated when the solution changes to blue
phytohormones. However, the potential of color (Syamsia et al., 2015). The absorbance
rhizome endophytic fungi in nutgrass as was analyzed using a UV-VIS
phosphate solvents and IAA producers has spectrophotometer with a wavelength of 840
never been analyzed and published. Aside from nm, while the data were processed using the
this analysis, molecular markers are also used formula y = 0.0271x + 0.0623 (Hutton &
to analyze endophytic fungal species based on Gregory, 1837). Moreover, the phosphate
the rDNA-Internal Transcribed Spacer (ITS) concentration was calculated based on the
sequences which located in 5-28S rDNA and standard phosphate curve in ppm units and the
the main evolutionary area. Furthermore, it is applied standard curve was KH2PO4 with a
also used in species-level comparison concentration of 0, 2.5, 5, 10, 20, and 50 ppm
(Bokulich & Mills, 2013; Elías-Román et al., (Lynn et al., 2013).
2018). Therefore, this study aims to analyze the Analysis of IAA production. Potato
potential of endophytic fungi in C. rotundus L. dextrose broth (PDB) medium was sterilized
as phosphate solvents and IAA producers. In using an autoclave with a pressure of 1 atm and
addition, the molecular analysis of the species 121°C for 15 min. The media were then added
found was also performed using rDNA-ITS with a precursor L-tryptophan solution (10 g
sequences. glucose, 1 g L-tryptophan, 0.1 g yeast extract
dissolved in 100 ml sterile water) in a ratio of
MATERIALS AND METHODS 20:1 for each tube. Furthermore, the endophytic
Isolation of endophytic fungi. C. fungi were inoculated in PDB at 25°C, and 160
rotundus L. rhizome was washed for 10 min rpm for 5 days, while the isolates were filtered
and then cut in a 2-3 cm (Hasyyati et al., 2017). using Whatman filter paper. The filtered
The samples were immersed in 75% alcohol for endophytic fungi colonies and PDB media were
1 min, 5.3% NaOCl for 3 min, and 75% alcohol centrifuged at 6000 rpm for 15 min, while 2 ml
solution for 30 s. Furthermore, the samples of the supernatant was taken, placed into a test
were dried using sterile tissue, split using a tube and then added with 4 ml of Salkowski
sterile knife, and placed in a Petri dish reagent (36.8 ml H2SO4 96%, 1.8 ml
containing PDA media (Widowati et al., 2016). FeCl3.6H2O 0.5 M, and 61.4 ml distilled water)
Analysis of phosphate solubilizing. The and incubated in a dark room for 60 min. A
obtained endophytic fungi isolates were positive result is indicated when the solution
cultured in 10 ml of Pikovskaya broth media changes to pink color. The IAA content was
with Ca5(PO4)3 as the phosphate inorganic analyzed using a UV-VIS spectrophotometer
source. The Pikovskaya broth medium contains with a wavelength of 540 nm. The blank
Ca5(PO4)3OH 5 g, glucose 13 g, (NH4)2SO4 0.5 solution used was PDB media added with L-
g, NaCl 0.2 g, FeSO4·7H2O 0.0002 g tryptophan, meanwhile, the concentration of
MgSO4·7H2O 0.1 g, yeast extract 0.5 g, KCl 0.2 IAA produced was determined using the
Vol 9(1), June 2021 Biogenesis: Jurnal Ilmiah Biologi 95

formula y = 0.0221x + 0.039 (Hutton & Analyzer version 2.0, while, the purity value
Gregory, 1837). Furthermore, the IAA was determined by calculating the absorbance
concentration was based on pure IAA in the ratio Å260/Å280. Meanwhile, DNA qualitative
standard curve of the sample. The applied analysis was determined using 0.8% agarose
concentrations include 0, 2.5, 5, 10, 20, and 50 gel electrophoresis (Tripathy et al., 2017).
ppm (Dewi et al., 2015). Amplification of Polymerase Chain
Morphological characterization. The Reaction (PCR). The PCR amplification was
morphological characteristics were analyzed by carried out using the molecular target in primer
macroscopic and microscopic observation. R-ITS1 (TCCGTAGGTGAACCTGCGG) and F-
Microscopic observations were performed ITS4 (TCCTCCGCTTATTGATGC) (Zhang et
using the basic slide culture method by (Rosana al., 2010). The composition of a total PCR
et al., 2014). Potato dextrose agar (PDA) volume is 25 µl according to (Geisen et al.,
medium was cut 0.5-by-0.5 cm block and 2017). Furthermore, PCR was carried out using
placed on the glass slide, meanwhile, the the tool, My CyclerTM Cycler BIO-RAD
inoculation was carried out using a sterile wire Thermo Cycler, while ITS1 and ITS4 rDNA
needle from the culture plate to four sides of the sequences amplification applied an annealing
PDA block. Sterile water was added to the temperature of 55°C. The PCR results were
tissue in the Petri plate, the cover was replaced, analyzed using 1.5% (v/v) agarose gel
and the basic slide culture was incubated at electrophoresis for 30 min with a voltage of 100
30°C. Observation on the morphological v, 400 mA. The gel from electrophoresis was
characteristics was based on an identification immersed in ethidium bromide (EtBr) for 15
guide book titled description of medical fungi minutes and was visualized using the Gel
(Kidd et al., 2016), pictorial atlas of soil and DocTM XR Imaging System BIO-RAD.
seed fungi, morphologies of cultured fungi and Sequencing analyses. The sequencing
key to species (Watanabe, 2010). result data were read using Sequence Scanner
DNA extraction and quantification. A 1.0 and compared to Gen Bank via the BLAST
total of 100 mg of endophytic fungi mycelium menu on NCBI. Furthermore, the alignment
aged 7 days was taken, crushed in a sterile was carried out using the Clustal-X program,
mortar, transferred into a 2 ml tube, added with while the data were imported directly to MEGA
1000 µl of 2x CTAB buffer, vortexed, and 5.0 for phylogenetic tree analysis. The
incubated in a water bath at 65°C for 60 min. phylogenetic tree was constructed using the
Furthermore, it was added with 900 µl of Neighbor-Joining algorithm with 1000
chloroform:isoamyl alcohol (24:1), incubated Bootsrap based on p-distance.
for 1 h at room temperature, and then Data analysis. The quantitative data on
centrifuged for 10 min at 13000 rpm. The dissolved phosphate levels and IAA were
supernatant was pipetted in a 1.5 ml tube, added analyzed using SPSS 16.0. In addition, the data
with 1x volume of chloroform:isoamyl alcohol were tested for normality using the Shapiro-
(24:1) and centrifuged for 10 min at 13000 rpm. Wilk and homogeneity using Levene’s test.
The precipitation stage was performed using When the data were normal and homogeneous,
isopropanol with a dose of 2/3 from the volume then it was followed by the ANOVA and
of the supernatant. The solution was left to DMRT test when there are differences in the
stand overnight at a temperature of -4°C, and effect of the isolates.
then centrifuged for 10 min at 13000 rpm.
Furthermore, the pellets were washed using 500 RESULTS AND DISCUSSION
µl of absolute ethanol and then centrifuged for Phosphate solubilizing levels produced
5 min at 13000 rpm. The samples were dried in by endophytic fungi. The endophytic fungi
an oven at 25°C and then added with 20 µl of obtained from the isolation and purification of
elution buffer and stored at -4°C (Aamir et al., nutgrass (C. rotundus L.) produced five isolates
2015). DNA quantification was conducted coded URT1, URT2, URT3, URT4, and URT5.
using nano drops AE-Nano200 Nucleic Acid The solubilizing phosphate in the endophytic
Kusmiyati et al. Biogenesis: Jurnal Ilmiah Biologi 96

fungi of nutgrass showed positive results f

Phosphate solubilizing level

100
indicated by the blue color with varying density 90 e
in each isolate (Fig. 1). 80 d
70 c
b

(ppm)
60
50
40
30
20
a
10
0
URT 1 URT 2 URT 3 URT 4 URT 5 Control
Fig. 1. The reaction of blue molybdenum with (-)
endophytic fungi isolates of nutgrass: (K-) negative Sample
control; (-) does not change in color; (+) light blue; (++)
Fig. 2. Dissolved phosphate levels of endophytic fungi of
dark blue; (+++) deep blue.
nutgrass. Different superscripts indicate the comparison
of means that are significantly different (p < 0.05).
Based on the results, URT4 produced the
strongest color, URT1 and URT2 were dark Indole-3-acetic acid levels produced by
blue, while, URT3 and URT5 produced a light endophytic fungi. Five isolates showed
blue color. Meanwhile, Pradhan & Pokhrel positive results in the test of IAA production
(2013) reported that the blue color density from (Fig. 3). The positive results were indicated by
the blue molybdenum reaction indicates the a change in the color of the supernatant solution
level of dissolved phosphate concentration from dark yellow to pink, meanwhile, this
produced by the sample. The blue color was solution was reacted with Salkowski’s reagent.
produced due to a chemical reaction. Lestari et al. (2015) reported that the formed
Phosphoric acid (dissolved phosphate) binds to IAA concentration is characterized by a change
ammonium molybdate and form heteropoly in the color of the supernatant solution reacted
acid which is then reduced by hydrazine sulfate with Salkowski’s reagent from yellow to pink.
to form phosphomolybdenum indicated by a In the negative control, no color change was
blue color solution. observed. This is because the L-tryptophan in
The quantitative analysis showed that the PDB media was not converted into IAA hence,
endophytic fungi of nutgrass produced dissolve the indole reaction was not achieved by
phosphate ranging from 54.03-87.83 ppm (Fig. Salkowski’s reagent. Dewi et al. (2015) stated
2). Furthermore, the statistical analysis that the higher the color density, the higher the
indicates that each isolate and negative controls IAA production. Based on the color of the
were significantly different (p<0.05). The solution formed, the highest qualitative IAA
highest dissolved phosphate level was produced levels were found in the URT1 isolate.
by the URT4 isolate, while the lowest was Furthermore, URT3, URT4, and URT5 showed
produced by the URT3. These results indicate a a change in color into pink, indicating that the
higher score compared to Syamsia et al. (2015), level of IAA production was moderate.
which reported that the endophytic fungi isolate Meanwhile, in the URT2 isolate, the reaction
of Enrekang aromatic rice produced dissolve color was yellow which indicates a low level of
phosphate ranging from 8.92-10.86 ppm. IAA production. The concentration of L-
Furthermore, the quantitative and qualitative tryptophan is presumably one of the factors that
analysis of the nutgrass endophytic fungi also determine the optimal level of IAA produced,
showed that the URT4 isolate had the highest meanwhile, the optimal level is determined by
ability to dissolve phosphate. the color density produced by each isolate.
Vol 9(1), June 2021 Biogenesis: Jurnal Ilmiah Biologi 97

60

IAA production level (ppm)

e
50
40
30 d
b c c
20
10 a
0
Fig. 3. The reaction of Salkowski’s reagent with URT 1 URT 2 URT 3 URT 4 URT 5 Control
endophytic fungi isolates of nutgrass: (K-) negative (-)
control; (-) does not change in color; (+) yellow; (++)
pink; (+++) deep red. Sample

The quantitative analysis showed that Fig. 4. The concentration of IAA production from
endophytic fungi of nutgrass. Different superscripts
endophytic fungi of nutgrass produced IAA. indicate the comparison of means that are significantly
Based on statistical analysis, the IAA level different (p < 0.05).
produced showed that each isolate (except
URT3 and URT5) and negative controls were Morphological characterization. Based
significantly different (p < 0.05) (Fig. 4), while on the results, URT4 and URT1 had high levels
URT3 and URT5 showed no significant in both dissolved phosphate and IAA
difference (p > 0.05). Furthermore, the URT4 production tests. Therefore, morphological
isolate produced the highest levels of IAA, observations focused on both isolates. The
which varied between 5.58-45.50 ppm. The fungal colony of URT1 and URT 4 have
highest IAA level was produced by the URT1 distinctive characteristics as presented in Fig. 5.
isolate, while the lowest was produced by the The colony was white with aerial hyphae
URT2. However, this result was also higher (URT1), zonate between brown and green with
compared to Mehmood et al. (2018) which floury (URT4), macroconidia 29.15 µm
stated that the IAA production of F. oxysporum (URT1), conidia 9-10 µm in diameter (URT4),
ranged from 1.5-2.5 mg/L. conidial head 235 x 163 µm in diameter.

a b c d

e f g h
Fig. 5. The morphological (colony appearance, conidial head, and macroconidia) from endophytic fungi of nutgrass: a.
URT1 (upper side); b. URT1 (reverse side); c. URT1 (macroconidia); d. URT1 (chlamydospore); e. URT4 (upper side);
f. URT4 (reverse side); g. URT4 (conidia); h. URT4 (conidial head).

Visualization of PCR. The visualization both isolates were parallel to the marker with a
of URT1 and URT4 PCR amplification is size of 550 and 625 bp respectively.
presented in Fig. 6. The results showed that
Kusmiyati et al. Biogenesis: Jurnal Ilmiah Biologi 98

Meanwhile, Poczai & Hyvönen (2010) showed isolates with other sequences in the NCBI was
that the ITS area varies between 500-750 bp. conducted using the BLAST program and the
results are presented in Table 1. The URT4
isolate had 97.42% similarity with A. terreus
AGE while URT1 had 100% similarity with F.
oxysporum AS13-D89.
Table 1. BLAST results of URT1 and URT4 isolates of
the endophytic fungi of nutgrass rhizome (C. rotundus
L.).
BLAST
Isolate
Similarity
code Species Seq. id.
(%)
Aspergillus
URT4 97.42 MT152989.1
terreus AGE
Fusarium
URT1 oxysporum 100 MN153518.1
AS13-D89

The reconstruction of relationships and

Fig. 6. Visualization of DNA bands from PCR
diversity of organisms based on the level of
amplification on 1.5% agarose gel: M= Marker; C (-)= similarity was analyzed using phylogenetic and
negative control. the results are shown in Fig. 7. This
reconstruction based on rDNA-ITS sequences
Sequencing and phylogenetic analysis. indicated that there were two clades with a
Data sequencing analysis for URT4 and URT1 bootstrap of 76% and 100%.

Fig. 7. The reconstruction of the phylogenetic tree for endophytic fungi of nutgrass isolates using the neighbor-joining
method and the bootstrap with 1000 replications.

The URT4 isolate had 97.42% similarity implies that A. terreus and F. oxysporum have
percentage with A. terreus and a genetic great potential to dissolve phosphate and
distance of 0.026, indicating a high similarity produce IAA. Aspergillus genus is one of the
value. Meanwhile, the URT1 isolate had 100% fungi that has been shown to effectively
similarity with F. oxysporum and a genetic dissolve phosphate (Srinivasan et al., 2012).
distance of 0.00. Furthermore, URT4 showed This is due to its ability to survive in conditions
similarities to A. terreus, while the URT1 of low humidity, extreme temperatures, and
showed similarities to F. oxysporum. This low pH since the growth of phosphate-
Vol 9(1), June 2021 Biogenesis: Jurnal Ilmiah Biologi 99

solubilizing microorganisms is influenced by https://doi.org/10.13057/psnmbi/m010220.

soil acidity (Fatmala et al., 2015). Besides, the Dor E, Hershenhorn J. 2013. Effect of low temperature
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and F. oxysporum belong to the same division Hanna JW, Ross-Davis AL, Kim MS, Klopfenstein
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