Preli

inary Screening of Antagonistic Fungal Endophytes

from Zingiberaceae

Anisa Lutfia

, Yurnaliza

and Erman Munir

Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Medan, Indonesia

Keywords: Endophyte, Fungi, Hutan Sibayak, North Sumatera, Zingiberaceae.

Abstract: Efforts to elaborate the role endophytes have been intensively studied from a different view point

for the last two decades. One of the most encouraging works is to find potential sources for novel

medicinal compounds. Preliminary screening of fungal endophytes possessing antagonistic

activity from rhizomes of Zingiberaceae has been conducted. The aim of this study is to collect

potential antagonistic fungal strains against representative microbial pathogens: Staphylococcus

aureus, Escherichia coli and Candida albicans. Antagonisms assay was performed by agar plug

method in dual culture plate assay. The study found thirty-nine (39) fungal strains collected from

rhizomes of five species of Zingiberaceae namely Alpinia sp., Amomum centrocephalum,

Elettaria sp., Etlingera sp. and Hedychium coronarium. Among all strains tested, 20 of them

were antagonists of S.aureus, 3 of E.coli and 1 of C.albicans. All antagonist strains showed

different degree of inhibitory activities which indicated the different nature of fungal endophytes

and their chemical properties.

1 INTRODUCTION

Zingiberaceae is a family of herbs that grow

abundantly in tropical to subtropical region with

center of divergence located in Southeast Asia. The

medicinal herbs covers about 1400 species around

the region, mainly from Peninsular Malaysia,

Indonesia, Brunei, Singapore, Philippines and New

Guinea (Pandey, 2001). In North Sumatera itself,

about 47 species of Zingiberaceae have been

reported while most of them were known to inhabit

Hutan Sibayak (Siregar, 2008). Notable species like

ginger and turmeric, has long been known as

potential therapy towards some illness and diseases

such as, digestive disorders, fever, cold, cough,

arthritis and muscle cramps (Ma, 2012). In scientific

reports, biological activities from the Zingiberaceae

compounds are proven to act as anti-inflammation,

anti-tumor, anti-apoptosis and antimicrobials

(Karuppiah, 2012).

On the other hand, microbial resistance is a big

challenge on medicinal view point. These ways of

finding may be achieved through the exploitation of

fungal endophytes to synthesize novel antibiotics,

especially the modified or somewhat similar with

Zingiberaceae compounds. The first step to study the

potential fungal endophytes is by isolating some

antagonistic strains from healthy and non-

symptomatic plant parts. Successful isolation of

fungal endophytes have been reported by several

researchers. Antagonistic Penicillium sp. has been

isolated from Curcuma longa, exhibiting inhibitory

activity against Pseudomonas aeruginosa and

Klebsiella pneumoniae (Rathod, 2013). Chemical

compounds produced by culture of Pestalotiopsis

vaccinii were known to be a newly discovered

natural products, solely synthesized by the strain

itself (Wang, 2014; Wang, 2017). As far literatures

has been surveyed, the study on the study on

exploitation of fungal endophytes from

Zingiberaceae is still limited. In this study, we

reported that rhizomes of representative species

from Zingiberaceae in North Sumatera, were

harbored by several strains of fungal endophytes.

The results of antagonistic activity towards tested

pathogens were distinct among fungal strains,

indicating different capability of strains in producing

antimicrobial compounds.

Lutﬁa, A., Yurnaliza, . and Munir, E.

Preliminary Screening of Antagonistic Fungal Endophytes from Zingiberaceae.

DOI: 10.5220/0010082300420046

In Proceedings of the International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches (ICOSTEERR 2018) - Research in Industry 4.0, pages

42-46

ISBN: 978-989-758-449-7

2 MATERIALS AND METHODS

2.1 Plant Materials

Samples of wild Zingiberaceae were collected during

exploration in Hutan Sibayak, located in Deli serdang

district, North Sumatera. Sampling were conducted

incidentally without considering any climate and

spatial factors. Plants anchoring to soils were dug up

and cut to separate its shoots and roots. The root parts

or rhizome were wrapped with paper and stored in

plastic bags. Duplicate samples were collected

separately to be authenticated by Herbarium

Medanese, Universitas Sumatera Utara for

identification. In laboratory, rhizomes were later cut

into smaller segments and composites were made by

pooling segments into one bulk sample for each

species of Zingiberaceae. The samples were then

used in isolation step.

2.2 Isolation of Fungal Endophytes

Step in isolating fungal endophytes were based on

previous report (Yurnaliza, 2014). Bulk samples from

each species of Zingiberaceae were washed with

tap water to remove remaining soil and dirt. The

samples were surface-sterilized by dipping in 75%

ethanol for 2 min, 5.3% NaOCl for 5 min and 75%

ethanol for 30 secs. The pieces were again washed

several times with sterile distilled water to remove

remaining solutions. Samples were dried on Whatman

filter paper and cut into 1–2 smaller pieces. The

pieces were placed on top of Potato Dextrose Agar

(Oxoid™) supplemented with chloramphenicol.

Plates were incubated in ambient condition for 3 days.

Any visible fungal growth from each pieces were

then sub-cultured onto new medium to preserve

the strains. Each fungal strains were differentiated

from their colony appearances.

2.3 Antagonisms Assay of Fungal

Endophytes

Pathogenic strains used in this study were:

Staphylococcus aureus ATCC® 29213™,

Escherichia coli ATCC® 25922™ and clinical

strain of Candida albicans. Both S. aureus and E.

coli were first grown in Nutrient Agar (NA) while C.

albicans in Potato Dextrose Agar (PDA) prior

antagonisms assay. Antagonisms assay were

performed based on agar plug method in dual culture

plate assay (Balouiri, 2016).

Direct colony suspensions from each pathogenic

strains were made by swabbing colonies into sterile

physiological saline solution (0.95% NaCl) to obtain

600

= 0.5. One mililitre of cell suspensions were

mixed with 15 mL molten PDA (45

C) medium,

supplemented with 1% (w/v) yeast extracts for

bacteria and 1% peptone (w/v) for C. albicans.

Molten agar medium were then plated to obtain

microbial lawns. Three plugs of aerial mycelium

from each fungal endophytes were placed on top of

medium. Plates were incubated for 2 days in ambient

condition. Clear zones around mycelial plugs

indicating antagonisms were measured using

standard caliper in millimetre unit (mm).

3 RESULTS AND DISCUSSIONS

The species list of Zingiberaceae found in this study

along with its fungal endophytic associates is

presented in Table 1. Previous report revealed that

there were eight genera of Zingiberaceae in Hutan

Sibayak that were: Amomum, Etlingera, Geocharis,

Geostachys, Globba, Hedychium, Hornstedtia and

Zingiber (Siregar, 2008). Although we just managed

to collect five genera in this study, here we reported

two new genera, Alpinia and Elettaria.

From the results, it can be seen that isolated

fungal endophytes were distinct to each species as

shown in Figure 1. Although the isolates were

seemed to be different among others, morphological

characters alone are not enough to identify species

level. Further molecular characterization is needed

to ensure the species identification.

In this study, we successfully isolated 39 fungal

strains with 30% of isolates were from H.

coronarium (13 isolates). Rhizome as being part of

plants’ food reserve is thought to be harbored by

various endophytes. Previous study showed that

between parts like leaf, petiole, stem, root,

adventitious root and rhizome used in isolation of

endophytic fungi from H. coronarium, the rhizome

showed the greatest diversity in number of

culturable fungal strains. Although some exceptions

can be found in other species of Zingiberaceae, that

was Zingiber officinale and Amomum siamense with

most recovered isolates were from petiole and

pseudostem, respectively, rather than from their

rhizomes (Bussaban, 2001; Uzma, 2016).

While several authors have reported the number

of fungal isolates from species of Zingiberaceae,

most of their reports

are about culturable

actinomycetes from genera Alpinia, Curcuma,

Hedychium and Zingiber (Taechowisan, 2003;

Taechowisan 2003; Taechowisan, 2008;

Krishnapura, 2015). Based on our knowing, this is

Preliminary Screening of Antagonistic Fungal Endophytes from Zingiberaceae

the first report on succesful isolation of fungal

endophytes from Elettaria. Molecular

identification is needed to confirm the possibility of

finding novel fungal strains from this species and

is our current concerns. Results will be published

elsewhere.

Table 1: Species list of Zingiberaceae and their fungal endophytic associates

Species

Isolate Code

umbe

Isolates

lpinia sp.

JRT 1A, JRT 1B, JRT 2A, JRT 2B, JRT 2C, JRT 3A

momum

centrocephalum

RL 1

, JRL 1B, JRL 2

RL 2B,

RL 2C,

RL 2D,

RL 3

JRL 3B , JRL 4A

tlingera sp.

JRN 1A, JRN 1B, JRN 1C, JRN 2A, JRN 3A, JRN 4A, JRN 4B 7

letta

ia s

RS 1

RS 1B,

RS 1C,

RS 2

RS 2B 5

edycium

coronarium

RD 1

RD 2

RD 2B,

RD 2C,

RD 2D,

RD 3

RE 1

JRE 1B, JRE 2A, JRE 2B, JRE 4 A, JRE 4B

Total

Figure 1: Representative colony images of isolated fungal endophytes from Zingiberaceae

Another approach to improve the number of

isolated endophytes from plant parts, is by varying

the medium composition. Endophytic bacteria

isolated from rhizomes of Curcuma zedoaria were

succesfully recovered through the use of four

isolation medium, mainly modified Nutrient Agar

(NA) and Water Yeast Extract Agar (WYEA). The

study also incorporated turmeric extracts itself into

the medium to induce growth of certain endophytic

strains (Krishnapura, 2014).

The results of antagonism assay of fungal

endophytes against three pathogens are presented in

Table 2. Twenty isolates or more than 50% inhibit

S. aureus with various zone measuring from 8.46

to 30.75 mm. Three isolates inhibit E. coli and one

inhibit C. albicans. Among all, JRN 4B enable to

inhibit all tested pathogens. From the results it is

indicated that fungal endophytes from

Zingiberaceae mostly synthesized compounds

effectively against gram positive bacteria. The

analyses of compounds produced by isolates are

now under investigation.

Although majority of tested fungal strains did not

show any inhibition zones in this study, especially to

E. coli and C. albicans, most of them were observed

to grow on top of microbial lawns. We assumed that

this might be some type of interaction or synergisms

between two competing microbes. Since we were

using agar plug method to exhibit inhibitory effect

towards pathogen, it is also possible that the fungal

strains secreted antimicrobial metabolites into its

agar medium. Based on our observation, future study

on evaluating antimicrobial activity of fungal strains

whether from the extracts and culture filtrates, may

support our assumption.

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

4 CONCLUSIONS

Five species of Zingiberaceae sampled from Hutan

Sibayak, North Sumatera are known to be

inhabited by fungal endophytes. Thirty nine

culturable fungal strains were succesfully isolated

from rhizomes of Alpinia, Amomum, Etlingera,

Elettaria and Hedychium. The study also

reported the first successful attempt on isolating

fungal endophytes from genus Elettaria. Each

fungal strains showed different degree of

antagonisms against S. aureus while most of them

did not show any inhibition against E. coli and C.

albicans. Confirmation on antimicrobial

metabolites secreted by fungal strains will be

considered in future study to evaluate possible

novel antimicrobial compounds.

Table 2:

Diameter of inhibition zones among tested fungal isolates

Code Isolate

ete

of Inhi

on Zone (m

)

S. au

eus E. coli

. albicans

JRS

1A 30,75 - -

1B - - -

1C 11,35 - -

2A - - -

2B - - -

JRD

1A 24,65 - -

2A - - -

2B 8,46 - -

2C 19,55 - -

2D 23,61 - -

3A 23,85 - -

JRE

1A - - -

1B 9,41 - -

2A - - -

2B - - -

4A - - -

4B 18,23 - -

JRT

1A 20,2 - -

1B - - -

2A 10,28 - -

2B 16,13 13,3 -

2C 13,91 - -

3A - - -

JRL

1A 25,2 - -

1B 13,5 - -

2A - - -

2B - - -

2C 20,11 19,23 -

2D 9,51 - -

3A - - -

3B 13,2 - -

4A - - -

JRN

1A - - -

1B - - -

1C - - -

2A 13,06 - -

3A - - -

4A - - -

4B 23,36 14,86 15,77

Preliminary Screening of Antagonistic Fungal Endophytes from Zingiberaceae

Figure 2: Representative images of antagonistic (Left) and non-antagonistic (Right)

ACKNOWLEDGEMENTS

The authors would like to express the highest gratitude

to Universitas Sumatera Utara, for funding this

research under scheme of Penelitian Guru Besar

TALENTA-USU year 2017–2018, contract Number:

427/UN5.2.3.1/PPM/KP-TALENTA USU/2018.

REFERENCES

Balouiri, M., Sadiki, M., Ibnsouda, S.K., 2016.

Methods for In Vitro Evaluating Antimicrobial

activity: A Review. Journal of Pharmaceutical

Analysis, Vol. 6, No. 2, 71–79.

Bussaban, B., Lumyong, S.,Lumyong, P., McKenzie,

E.H., Hyde, K.D., 2001. Endophytic Fungi from

Amomum siamense. Canadian Journal of

Microbiology, Vol. 47, No. 10, 943–948.

Karuppiah, P., Rajaram, S., 2012. Antibacterial effect of

Allium sativum cloves and Zingiber officinale rhizomes

against multiple-drug resistant clinical pathogens.

Asian Pac J Trop Biomed, Vol. 2, No. 8, 597–601.

Krishnapura, P.R., Belur, P.D., 2015. Isolation and

Screning of Endophytes from the rhizomes of Some

Zingiberaceae Plant for L- Asparaginase

production. Preparative Biochemistry and

Biotechnology Vol. 46, No. 3, 281–287.

Ma, X., Gang, D.R., 2006. Metabolic Profiling of in vitro

Micropropagated and conventionally Greenhous

Grown Ginger (Zingiber officinale).

Phytochemistry, Vol. 67, No. 20, 2239–2255.

Pandey, B.P., 2001. A Text Book of Botany.

Angiosperms: Taxonomy, Anatomy,

Embryology, S.

Chand & Company. India,

Edition.

Rathod, V., Singh, D., Ninganagouda, S., Hiremath, J.,

Kulkarni, P., 2013. Biosynthesis Of Silver Nanoparticle

By Endophytic Fungi

Penicillium

sp. Isolated From

Curcuma longa

(turmeric) And Its Antibacterial

Activity Against Pathogenic Gram Negative Bacteria.

Journal of Pharmacy research,

Vol.

448–453.

Siregar, E.S., Pasaribu, N., 2008. Inventarisasi Jenis-

JenisZingiberaceae di Hutan Sibayak

Sumatera Utara.

Jurnal Penelitian MIPA

Vol.2, No. 1,

22–24.

Sulistiyani, T.R., Lisdiyanti, P., Lestari, Y., 2014.

Population and Diversity of Endophytic Bacteria

Associated with Medical Plant

Curcuma zedoaria

Microbiology Indonesia,

Vol. 8, No. 2, 65–72.

Taechowisan, T., Peberdy, J.F., Lumyong, S., 2003.

Isolation of Endophytic actinomycetes from selected

plants and their antifungal activity.

World Journal

of Microbiology and Biotechnology,

Vol. 19, No. 4,

381–385.

Taechowisan, T., Lumyong, S., 2003. Activity Of

Endophytic Actinomycetes From Roots Of

Zingiber

officinale

and

Alpinia galanga

Against

Phytopathogenic Fungi.

Annals of Microbiology,

Vol.

53, No. 3, 291–298.

Taechowisan, T., Chuaychot, N., Chanaphat, S.,

Wanbanjob, A., Shen, Y.,

et al

2008. Biological

Activity of Chemical Constituents Isolated From

Streptomyces

sp. Tc025, an Endophyte in

Alpinia

galanga. International Journal of Pharmacology,

Vol.

4, No. 2

95–101.

Uzma, F., Konappa, N.M., Chowdappa, S., 2016.

Diversity and Extracellular Enzyme Activites ofM

Fungal

Endophytes

Isolated from Medicinal

plants of Western Ghats, Karnataka.

Egyptian

Journal of Basic and Applied Sciences,

Vol. 3, No. 4,

335–342.

Wang, J., Wei, X., Lu, X., Xu, F., Wan, J., Lin, X., Zhou,

X., Liao, S., Yang, B., Tu, Z., Liu, Y., 2014. Eight

New Polyketide Metabolites From the Fungus

Pestalotiopsis vaccinia

endogenus with the Mangrove

Plant

Kandelia candel

(L) Druce.

Tetrahedron,

Vol.

70, No. 51, 9695–9701.

Wang, J.F., Liang, R., Liao, S.R., Yang, B., Tu, Z.C.,

Lin, X.P., Wang, B.G., Liu, Y., 2017. Vaccinols J-S, ten

new Salicyloid Derivatives from the Marine Mangrove

Derived Endophytic Fungus

Pestalotiopsis vaccinii

Fitoterapia,

Vol. 120, 164–170.

Yurnaliza, Aryantha, I.P.G., Esyanti, R.R., Susanto, A.,

2014. Antagonistic Activity Assessment of Fungal

Endophytes From Oil Palm Tissue Against

Ganoderma

boninense

pat.

Plant Pathology Journal

, Vol. 13, No. 4,

257–267.

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches