340 Yadanabon University Research Journal 2022, Vol.12, No.

Screening of Endophytic Fungi from Hypselandra variabilis ( Coll & Hems )

Pax & Hoffm and their Starch Hydrolysis Activities
Hla Myo Thein*, Khin Thida Swe** , Swe Zin Win***

Abstract
The endophytic fungi were isolated from various parts of Hypselandra variabilis (Coll &
Hems) Pax & Hoffm (Thamon) plant grown at Tha-Yet- Kan village, Madayar Township,
Mandalay Region, during July to September 2021. The endophytic fungi were isolated by
surface sterilization method (NITE, 2004). A total fifteen fungi were isolated according to
their morphological characters. The amylase enzyme activities of fungal isolates were
detected by method of NITE, 2004. The pure endophytic fungi were tested by iodine for
the study of starch hydrolyzing activities. Among them, eleven endophytic fungi showed
the starch hydrolyzing activities. And then, pure isolated fungi were inoculated into test
tubes containing isolation medium for the further research such as antimicrobial activities.
Keywords: endophytic fungi, starch hydrolyzing activities

Introduction
Endophytes are microbes existing within the plants without causing any disturb to their
growth and development. Unlike disease-causing microorganisms, endophytes are non-
pathogenic and many of them are known to promote their plant host's fitness (Mendes et al.,
2013). In recent years, it is gradually conceded that endophytic fungi or endophytes have played
a very important role in affecting the quality and quantity of the crude drug through a particular
fungus-host interaction, indicating that more appreciating on the particular relationships
between endophytic fungi and medicinal plants is required for promoting crude drug production
(Faeth and Fagan, 2002).
Hypselandra variabilis (Coll & Hems) Pax & Hoffm is a tropical species belongs to family
Capparaceae. It is indigenous to Myanmar and its local name is Thamon. The leaves and roots are
used in aching, sedema, cold extremities and also used as stomachic, expectorant and counter
irritant. Fruits and vegetables are rich in minerals, vitamins and fibre and low in saturated fat
(Batta, 2016). Since this plant is consumed as seasonal food by local people in Myanmar, it is
required to examine the nutritional and medicinal value of this plant (San and Han, 1998). The
crown gall of the Thamon is used to relieve eye sore. It is believed that eating Tha-Mon once a

*
Lecturer, Department of Botany, Yadanabon University
**
Lecturer, Dr, Department of Botany, Yadanabon University
***
Associate Professor, Dr, Department of Botany, Yadanabon University
Yadanabon University Research Journal 2022, Vol.12, No.2 341

year can keep the good health along the lifespan. Wild edible plants are still consumed
traditionally by different coummunities mainly in rural and suburban areas (Pinela et al., 2017).
Thamon flowers contain a number of beneficial antioxidants including polyphenols.
Antioxidants against and free radicals, which are by products of cell oxidation. Free radicals are
associated with causing a number of health problems, including breast, prostate and lung
cancers (Bhowmik and Lombardi, 2014).
In the present study, an attempt has been made to offer an endophytic fungus as source
of enzymes for industrial essential. The aim of the present study was to screen the different
endophytic fungi for amylase production isolated from Hypselandra variabilis (Coll & Hems) Pax
& Hoffm (Thamon) plant. The isolated endophytic fungi will have antimicrobial activities in a later
article and will synthesize compound the best of them.

Materials and Methods

Collection of Plant Sample
The Hypselandra variabilis (Coll & Hems) Pax & Hoffm (Thamon) plants were collected
from Tha-Yet-Kan Village, Madayar Township, Mandalay Region(N 22º 4´21'' and E 96º 6´31'').
Sample collection was carried out in July to September 2021. Disinfected plant samples were
collected and sealed in the polyethylene bags. The sample preparations were performed at
Microbiology laboratory of Botany Department, Yadanabon University.

Collected Area

Figure.1 Map of Madayar Township

(Source: Google Map)
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Identification of Plant Sample

The collected plant samples were identified according to the reference books
(Backer and Bakhuizen Van Deen Brink, 1963) and (Dassanayake, 1980).
Isolation of Endophytic Fungi (Ando, et al., 2004)
The surface sterilization method (NITE, 2004) was used for the isolation of endophytic
fungi. The endophytic fungi were isolated from leaves, stems and flowers of the Hypselandra
variabilis (Coll & Hems) Pax & Hoffm (Thamon) plant. Plant sample was rinsed carefully in tap
water for 15 mins and air dried before it was processed. The plant materials were then surface
sterilized by soaking them consecutively in 70% ethanol for 60 seconds and then, also soaked
10% sodium hypochloride for 60 seconds and rinsed carefully with sterile distilled water. Then,
with a sterile scissors, outer tissue was removed and the inner tissues of 0.5 cm size were
delicately cut and placed on Czapek–Doz Agar (CZA) medium (Sucrose 3.0 g, NaNo3 0.2 g, K2HPO4
0.1 g, MgSO47H2O 0.05 g, KCL 0.05 g, FeSO47H2O 0.001 g, Agar 1.8 g, Distilled water 100 mL)and
Glucose Yeast Extract Peptone Agar(GYA)medium(Glucose 1.0 g, Yeast Extract 0.3 g, Peptone 0.3g,
K2HPO4 0.001 g, MgSO47H2O 0.001 g, Agar 1.8 g, Distilled water 100 mL). After autoclaving, the
medium was added with chloramphenicol to suppress bacterial growth. The Petri-disc was
incubated at room temperature for 3 days to 7 days until fungal growth appeared.
Starch hydrolyzing activities of Isolated Fungi (NITE, 2004)
The liquid medium (Soluble Starch 1.0 g, KH2 PO4 0.1 g, MgSO4 7H2O 0.1 g, NaCl 0.1 g, (
NH4 )2SO4 0.2 g, CaCO3 0.1 g and distilled water 100 mL) was prepared for the amylase activity
test according to the given composition and sterilized. Then about 20 mL of this medium was
poured into each test tube and autoclaved at 121ºC. After sterilization, these test tubes were
cooled at room temperature. The isolated fungi were inoculated into each test tube
respectively and incubated for 7 days. Then, Iodine solution (drop by drop 0.1 mL to 0.3mL)
was poured gently into each test tube containing liquid culture medium.
After adding the iodine solution, if the liquid culture medium changed to blue black
colour, it indicates that the isolated fungi cannot hydrolyse the starch. If the blue black colour
changed into pale colour or colourless, the isolated fungi can hydrolyse the starch.

Results
Identification of Plant Sample
Hypselandra variabilis (Collect & Hems) Pax & Hoffm
Local name : Thamon
English name : Unknown
Flowering period : January to March
Yadanabon University Research Journal 2022, Vol.12, No.2 343

Perennial unarmed shrubs; stems and branches terete, pubescent when young. Leaves
palmately compound, alternate, stipulate; leaflet 3-5, obovate-oblong, petiolate. Inflorescences
terminal or axillary, corymbose cymes, flowers bisexual, actinomorphic, tetramerous,
hypogynous, creamy-white, about 1.5 cm in diameter at anthesis, apetalous, bisexual, bracts
leafy, subtending the flowers. Calyx campanulate, deeply 4 partite. Disc fleshy, green, surrounding
the androgynophore. Stamens 8-10, free, exserted; filaments filiform, white; anthers ovoid. Ovary
superior, ellipsoid, unilocular, with many ovules on the parietal placentae; stigma discoid.
Baccate ovoid, glabrous; seeds fleshy.
Distribution : This species was recorded in Myanmar (Kress et al. 2003).

Habit Leaf L.S of flower T.S of ovary

Figure.2 Morphological Characters of Hypselandra variabilis (Collect & Hems) Pax & Hoffm
Isolation of Endophytic Fungi
Fifteen endophytic fungi were isolated from leaves, stems and flowers of Thamon plant.
Pure culture of isolated endophytic fungi were named as HMO-01 to HMO-15 (Figure - 3 to 14).
Table.1 List of Endophytic Fungi Isolated from parts of Hypselandra variabilis ( Coll & Hems )
Pax & Hoffm (Thamon)
Media Plant Parts Fungi Number Isolated Fungi
GYA Leaves HMO – 01, 02, 03 3
GYA Stems HMO- 04, 05 2
GYA Flowers HMO- 06, 07 2
CZA Leaves HMO-08, 09,10 3
CZA Stems HMO-11,12,13 3
CZA Flowers HMO- 14, 15 2
Total isolated fungi 15

Surface view Reverse view Photomicroscopy X 40

Figure.3 Colony morphology and photomicroscopy of endophytic fungus HMO-01
344 Yadanabon University Research Journal 2022, Vol.12, No.2

Surface view Reverse view Photomicroscopy X 40

Figure.4 Colony morphology and photomicroscopy of endophytic fungus HMO-02

Surface view Reverse view Photomicroscopy X 40

Figure.5 Colony morphology and photomicroscopy of endophytic fungus HMO-03

Surface view Reverse view Photomicroscopy X 40

Figure.6 Colony morphology and photomicroscopy of endophytic fungus HMO-04

Surface view Reverse view Photomicroscopy X 40

Figure.7 Colony morphology and photomicroscopy of endophytic fungus HMO-05

Surface view Reverse view Photomicroscopy X 40

Figure.8 Colony morphology and photomicroscopy of endophytic fungus HMO- 06
Yadanabon University Research Journal 2022, Vol.12, No.2 345

Surface view Reverse view Photomicroscopy X 40

Figure.9 Colony morphology and photomicroscopy of endophytic fungus HMO-07

Surface view Reverse view Photomicroscopy X 40

Figure.10 Colony morphology and photomicroscopy of endophytic fungus HMO-08

Surface view Reverse view Photomicroscopy X 40

Figure.11 Colony morphology and photomicroscopy of endophytic fungus HMO-09

Surface view Reverse view Photomicroscopy X 40

Figure.12 Colony morphology and photomicroscopy of endophytic fungus HMO-10

Surface view Reverse view Photomicroscopy X 40

Figure.13 Colony morphology and photomicroscopy of endophytic fungus HMO-11
346 Yadanabon University Research Journal 2022, Vol.12, No.2

Surface view Reverse view Photomicroscopy X 40

Figure.14 Colony morphology and photomicroscopy of endophytic fungus HMO-
12

Surface view Reverse view Photomicroscopy X 40

Figure.15 Colony morphology and photomicroscopy of endophytic fungus HMO-13

Surface view Reverse view Photomicroscopy X 40

Figure.16 Colony morphology and photomicroscopy of endophytic fungus HMO-14

Surface view Reverse view Photomicroscopy X 40

Figure.17 Colony morphology and photomicroscopy of endophytic fungus HMO-15
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Table.2 Morphological and Microscopical Characters of Endophytic Fungi

Morphological characters Microscopical characters
Strain No. Surface view Reverse view Size (5 Days Shape Conidia Media
old culture)
Yellowish pink colour in the 7.3cm
HMO – 01 White colour Spindle Many GYA
center and edge white colour
Brownish gray in the
Dark brownish gray in the
HMO – 02 center and edge white 6.3 cm Spherical Many GYA
center and edge white colour
colour
Yellowish pink colour in the
HMO – 03 White colour 8.6 cm Spindle 2 GYA
center and edge white colour
Light Gray in the center Dark Gray in the center and Ovoid to short
HMO – 04 5.6 cm 4 GYA
and edge white colour edge white colour cylindrical
Pale Gray in the center and Antique bronze in the center Ovoid to short
HMO – 05 6.3 cm 3 GYA
edge white colour and edge white colour cylindrical
Pale black in the center and
HMO – 06 Pale brown colour 6.3 cm Spindle Many GYA
edge yellowish pink colour
Black in the center and edge Globose Singly or
HMO – 07 Cream colour 3.7 cm GYA
white colour phialides in groups

HMO – 08 Cream colour Pale pink 5.9 cm Ovate -oblong 2 CZA

Brownish gray in the

Pale black in the center and
HMO – 09 center and edge pale gray 6.0 cm Globose 1 CZA
edge white colour
colour
Olive colour in the center
Olive colour in the center
HMO – 10 and edge yellowish pink 5.0 cm Ovoid -oblong 1 CZA
and edge creamy colour
colour
Antique bronze in the center
HMO – 11 Antique bronze colour and edge pale Antique 6.0 cm Globose 1 CZA
bronze colour
HMO – 12 White colour White colour 7.3 cm Crescent 1- 3 CZA
HMO – 13 Neutral gray colour Pale Neutral gray colour 7.3 cm Spherical many CZA
Globose Singly or
HMO – 14 Titanium white Titanium white 9.0 cm CZA
phialides in groups
Creamy colour in the
Creamy colour in the center Ovoid to short
HMO – 15 center and titanium white 6.5 cm 5 CZA
and titanium white colour cylindrical
colour

Starch hydrolyzing activities of Isolated Fungi (NITE, 2004)

In the screening of amylase enzymes from isolated endophytic fungi were carried out,
eleven endophytic fungi (HMO-02, HMO-03, HMO-05, HMO-06, HMO-09, HMO-10, HMO-11, HMO-12,
348 Yadanabon University Research Journal 2022, Vol.12, No.2

HMO-13, HMO-14 and HMO-15) showed starch hydrolyzing activities. The rest four fungi (HMO-
01, HMO-04, HMO-07, HMO-08) do not show the starch hydrolyzing activities(Figure-18).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Figure.18 Starch hydrolyzing activity of endophytic fungi HMO-01 to HMO-15

Discussion and Conclusion
Studies have shown that some medicinal properties of plants may be related to their
endophytic fungi (Zou et al., 2000). Endophytic fungi have been explored from various plant
sources for their industrial potential. Therefore researchers focused on terrestrial plants for
endophytic (Bindu et al., 2021).
In the present study, an attempt has been made to offer an endophytic fungus as source
of enzymes for industrial requirements. A total of fifteen endophytic fungal strains were screened
from leaves, stems and flowers of Hypselandra variabilis (Coll & Hems) Pax & Hoffm (Thamon)
inoculated on Glucose Yeast extract Agar (GYA) and Czapek- Dox Agar (CZA) media. In this study,
HMO-01, HMO-03, HMO-12, HMO-13, and HMO-14 showed excellent growth on PGA (Potato
Glucose Agar) medium. However, HMO-02, HMO-04, HMO-05, HMO-06, HMO-08, HMO-09, HMO-10,
HMO-11 and HMO-15 showed moderate growth and only HMO-07 showed to be the least (poor)
growth on PGA medium (Table-2).
According to the results of starch hydrolyzing activities, these eleven endophytic fungal
strains (HMO-02, HMO-03, HMO-05, HMO-06, HMO-09, HMO-10, HMO-11, HMO-12, HMO-13, HMO-14
and HMO-15 ) changed into pale colour or colourless, therefore, they showed the starch
hydrolyzing activities (Figure-18). However, HMO-01, HMO-04, HMO-07 and HMO-08 showed blue
black colour, it indicates that the isolated endophytic fungi cannot hydrolyse the starch
(Figure-18).
It was concluded that the medicinal plant Hypselandra variabilis (Coll & Hems) Pax &
Hoffm (Thamon) has numerous types of endophytic fungi. The isolated endophytic fungi will be
tested antimicrobial activities in a later article. Among them, a strain that exhibit the highest
antimicrobial activity will be selected for further study.
Yadanabon University Research Journal 2022, Vol.12, No.2 349

Acknowledgements
First of all, we intend to declare our gratefulness to Rector and Prorectors, Yadanabon University,
for their permission us to use the research laboratory at Yadanarbon University. We are stretch my sincere
thanks to Professor and Head, Department of Botany, Yadanabon University, for her permission to do this
research and for supporting departmental facilities. We are also grateful to Professors and associate
Professors, Botany Department Yadanabon University for their incitement to write this research paper.

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