Jurnal Agrisistem, Juni 2009, Vol. 5 No.

ISSN 1858-4330

ISOLATION OF ENDOPHYTIC ACTINOMYCETES USING SURFACE

STERILIZATION METHOD AND SELECTIVELY ISOLATION MEDIA
ISOLASI ACTINOMYCETES ENDOFITIK MELALUI METODE STERILISASI
PERMUKAAN DAN ISOLASI MEDIA TERSELEKSI
Sitti Inderiati
Estate Crops Cultivation Study Program, Pangkep State Polytechnic of Agriculture
Pangkajene dan Kepulauan Regency, South Sulawesi

ABSTRACT
Cultivation based method is commonly used to isolate endophytic actinomycetes from
plant cells. To survey endophytic actinomycetes as potential biological control agents
against phytopathogens, young plants of tomato were surfaced-sterilized for use an
isolation source. In the present study, 36 endophytic actinomycetes were isolated from
roots, stems and leaves of healthy tomato plants. Of these, isolates recovered from roots
were more diverse than from leaves and stems. The isolates were identified by using 16S
ribosomal RNA (rRNA) gene sequencing and found to belong to a small group of
actinomyecetes genera including Streptomyces, Microbispora, and Nonomurae sp. which is
first time found as endophyte. On the basis of isolation media used, HV agar containing
vitamin B and humic acid as the sole carbon and nitrogen was more efficient to other
currently used media, including Tap Water Yeast Extract and Casamino acid media.
Key words: actinomycetes, endophytic, sterilization

ABSTRAK
Metode untuk mengisolasi endofitik actinomycetes dari tanaman inang telah
dikembangkan melalui sterilisasi permukaan dengan beberapa media untuk inkubasi.
Untuk mendapatkan endophytic actinomycetes yang berpotensi sebagai agens hayati
terhadap patogen, tanaman tomat pada fase vegetatif aktif dijadikan sebagai bahan isolasi
menggunakan metode sterilisasi permukaan. Pada penelitian ini, diperoleh 36 jenis
endophytic actinomycetes dari akar, batang, dan daun tanaman tomat. Dari ketiga bagian
tersebut, mayoritas isolat berasal dari bagian akar dan hanya sebagian kecil berhasil
diisolasi dari bagian lainnya. Isolat diidentifikasi menggunakan metode 16S ribosomal
DNA (rDNA) gen sekuens dan teridentifikasi sebagai bagian kecil dari genus
Streptomyces, Microbispora, and Nonomurae sp. Yang untuk pertama kalinya ditemukan
sebagai endofitik. Berdasarkan media isolasi yang digunakan, medium HV agar yang
mengandung asam humik sebagai sumber utama karbon dan nitrogen memberikan hasil
isolasi terbanyak dibanding dengan kedua media isolasi lainnya, yaitu media Tap Water
Yeast Extract dan Casamino Acid.
Kata kunci: actinomycetes, endofitik, sterilisasi

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INTRODUCTION
Bacterial endophytic species are present in
a wide range of plant species and reside
either within cells, in the intracellular
space, or in the vascular system of a plant.
Microbial endophytic are typically defined
as microorganisms that are detected after
surface sterilization of a plant part and
cause an apparent, asymptomatic infection
in healthy plant tissue but cause no
symptoms of disease. The study of plantassociated bacteria is important to understand their ecological role and interaction
with plants and for biotechnological
applications, such as biological control of
plant pathogens and isolation of valuable
compounds.
Although it has been known for long time,
their significance become evident only
more recently when it was shown that
they play specific roles as for instance,
protecting the plant hosts against phytopatogens. Among the bacterial endophytes, actinomycetes have received considerably attention due to their long time
prominence as the main source of wide
spectrum of antibiotics and other beneficial compounds of commercial interest.
Some actinomycetes are found to colonize
the interior of healthy plants; Frankia
strains are simbiotans of actinorhizal
plants, can induce N2-fixing root nodules
on certain nonleguminous plants and were
identified as actinomycetes in 1964
(Benson & Silvester 1993 in Cao et al.
2004a). To date, endophytic actinomycetes have been successfully isolated from
a wide variety of plants, such as rhododendron (Shimizu et al. 2000), wheat
(Coombs and Franco 2003), rice (Tian et
al. 2003), banana (Cao et al. 2004a), lupin
(El-Tarabily 2003) and tomato (Cao et al.
2004b). Taechowisan et al. (2002) isolated
330 strains of actinomycetes from leaves,
stems and roots of 36 plant species.
As endophytes, the isolate actinomycetes
colonize the same niche as plant patho-

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gens and therefore may be better acclimatized to the plant than rhizosphere
bacteria to control pathogens. Endophytic
actinomycetes have been shown to have a
number of beneficial effects on the host
plant when reintroduced. Actinomycetes
endophytes are reported to control fungal
infection and accelerate plant growth of
wheat (Coombs and Franco 2003), and
showed antagonistic activity against
Fusarium oxysporum f. sp. cubense (Cao
et al. 2004a).
With certain excellent features of these
gram positive bacteria, their isolation is an
important step for screening of new bioactive compounds. Therefore, this study
was designed to develop an effective method on isolation of endophytic actinomycetes from a horticultural plant and
evaluate the highly effective media for
cultivation-dependent approach.

MATERIALS AND METHODS

Sample collection
Seven week-old of healthy tomato plants
were collected from tomato field plots in
Murray Bridge, South Australia. A total of
nine plants were dug out carefully to
ensure that the intact root system was
removed and maximal amount of root
materials were collected.
Surface sterilization and isolation of
actinomycetes from tomato plants
The plants were thoroughly washed in
running tap water to remove all soil from
plant materials and then dissected into
roots, stems and leaves before being
subjected to a three-step surface sterilization procedure. The plant segments
were immersed in 99% ethanol for 60
seconds, followed by a 12 minute immerse in 6% NaOCl and a 60 second wash
in 99% ethanol. The surface-sterilized
plant segments were rinsed in sterile

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Jurnal Agrisistem, Juni 2009, Vol. 5 No. 1

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Reverse Osmosis (RO) water and aseptically sectioned into 1 cm long fragments.
Finally, the plant fragments were distributed onto the isolation media and
incubated at 27oC for up to 4 weeks. The
plates were then observed for the presence
of endophytic actinomycete colonies. The
colonies were then picked off using a
sterile needle and streaked onto subcultured media: a half strength Potato
Dextrose Agar (PDA) and Mannitol Soya
flour agar (MS) plates. Actinomycetes
growing on the media were purified and
identified.

doxine-HCl, inositol, Ca-pantothenate, paminobenzoic acid, and 0.25 mg of

biotion) was added on to autoclaved HV
medium. The last isolation medium was
Yeast Extract Casamino Acid (YECD;
containing 0.3g of yeast extract, 0.3g of
D-glucose, 2g of K2HPO4, 1g of Casamino
acid per liter of RO water).

RESULT AND DISCUSSION

Result
A total of 36 actinomycete strains were
isolated from healthy tomato plants, 20
strains were isolated from roots, 6 and 10
isolates were recovered from stem and
leave parts respectively. There were three
different media used as isolation media
and the percentage of effectiveness of
each medium was calculated by dividing
the total number of endophyte strains
isolated by the number of isolation plates
used. The effectiveness of isolation media
is shown in Figure 1.

Media Isolation
There were three different media used as
isolation media, as follows: Tap Water
Yeast Extract (TWYE; containing 0.25g
of yeast extract [Oxoid] , 0.5g of K2HPO4,
and 18g of Bacto agar [Oxoid] per litre of
tap water). Humic acid Vitamin C (HV;
containing humic acid 1.0g, Na2HPO4
0.5g, KCl 1.71g, MgSO4.7H2O 0.05g,
FeSO4.7H2O 0.01g, CaCO3 0.02g, bacto
agar 18g in 1000 ml of RO water). 0.5ml
of filter sterilized B-vitamins (0.5 mg each
of thiamine-HCl, riboflavin, niacin, pyri-

The isolates are most prevalent from roots

(56.66%), leaves (26.66%) and less from
stems (16.66%).

The percentage of effectiveness of each

isolation medium
58.33%
0.6
0.5

36.11%

0.4
0.3
0.2
5.50%

0.1
0
TWYE

HV

Figure 1. Percentage of effectiveness of isolation media

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YECD

Jurnal Agrisistem, Juni 2009, Vol. 5 No. 1

Table 1 illustrates the number of actinomycete endophytes isolated from roots,

stems and leaves of tomato plants. HV and
TWYE media are more effective for the
isolation of endophytic actinomyetes from
tomato plants as compared to YCED.
Approximately 58% and 36% of isolates

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were recovered on HV and TWYE media

respectively, and only small amount (5%)
were recovered from YECD medium. The
effectiveness of HV and TWYE media are
58.33% and 36.11% respectively while
YCED is 5.50%.

Figure 2. Endophytic actinomycete (arrow signs) emerged from roots and stems after 10
days of incubation on HV and TWYE media.

Table 1. Number of endophytic actinobacteria isolated from roots, stems and leaves of
tomato plants
Plant part

Number of isolates

Percentage (%)

Roots
Stems
Leaves

17
5
8

56.66
16.66
26.66

All isolates were first identified morphologically. 36 different isolates based on

morphology characterization were then
subjected to 16S ribosomal gene sequencing (identification result data not shown).
The sequences were compared with the
16S rRNA data stored in the GenBank of

NCBI. Analysis of these 16S rRNA genes

by BLASTN confirmed that the strains
isolated most frequently would be a
member of the genus streptomyces
(57.14%). Ten isolates (22.85%) were placed in the genus Microbispora according
to the closest match sequences found in
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Jurnal Agrisistem, Juni 2009, Vol. 5 No. 1

the databases and one isolate is most

closely related to the genus Nonomurae
sp. (2.85%) while the remaining were
unidentified (17.14%) but.
Discussion
In the present study, it has been showed
that the surface sterilization protocol is
effective in removing surface-adhering
microorganisms, including spore-bearing
actinomycetes, and that the isolates obtained can be considered to be true endophytes. The actinomycete isolates took at
least 3 weeks to grow out from the plant
tissues. If the tissue sterilization procedure
used in this study was not sufficient to kill
surface microbes, they would be expected
to grow from specimens within a few days
and could over-grow the plate and mask
the presence of the actinomycetes. Therefore, it is acknowledged that the actinomycetes isolated are true endophytes of
tomato plants.
It was recognized that media used for
isolation can contribute to the number and
variety of isolates obtained. HV agar
medium that containing humic acid as the
sole source of carbon and nitrogen produced higher numbers of actinomycete
colonies than the two other media. The
superiority of HV agar is related to the
special activity of actinomycetes in natural environment and to the property of
humic acid which activate actinomycetes
spores upon germination (Hayakawa and
Nonomura 1987). On the HV agar, Actinomycetes colonies developed well and
formed spores or sporangia quite abundantly from the aerial or substrate mycelia. Another medium in which actinomycete colonies appear relatively abundant was TWYE agar. This low nutrient
medium can retarded the growth of fungal
and eubacteria sufficiently to allow incubation of plates for 7 to 14 days, by which
time actinomycete colonies grew and
sporulated (Figure 2). The present of the
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contaminated microorganisms on isolation

plates may well inhibit and disguise the
growth of endophytic actinomycetes.
Based on that, the two isolation media
seem to be more functional to isolate
endophytic actino-mycetes, on that not
only streptomyces colonies recovered but
also other actinomycetes belonging to
Microbispora and Nonomurae emerged
and developed fairly well, whereas the
development of true bacteria and fungi
was restricted (Figure 2).
Analysis of the frequency of isolation of
endophytes from tomato plant indicates
that actinomycete isolates from roots were
more diverse than from stems and leaves.
Even though caution has to be exercised
due to the small sample size, these results
are consistent with the findings of
Coombs (2001), Taechowisan et al.
(2003), Tien et al. (2004), and Cao et al.
(2004). The relatively abundant diversity
in root is possible due to direct contact
with the soil (Tian et al. 2004), from
which actinomycetes endophytes colonize
the plant. Another interesting observation
is that the endophyte isolates had been
previously found to have an association
with other plants. Isolates belonging to the
genera Streptomyces and Microbispora
were found to be widely distributed
among plants species. Streptomycetes
were the most common isolates recovered
from 28 plant species (Sardi et al. 1992)
and from 36 distinct varieties of plants
(Taechowisan et al. 2003), while Okazaki
et al. (1995) suggested that Microbispora
spp. were more likely to be recovered
from plant tissues than from soil. The
large number of Streptomyces and
Microbispora isolated from healthy plants
indicates that there is a close association
between these microorganisms and plants,
in which growth of the actinomycetes
could have beneficial effects.
The isolation of microorganisms from
within the tissue of healthy tomato sug-

Jurnal Agrisistem, Juni 2009, Vol. 5 No. 1

gests that the host derives some benefit

from harboring the endophyte. In this
case, the advantage may take the form of a
secondary metabolite produced by the
endophyte, since actinomycetes are well
known for their ability to produce a broad
range of antimicrobial and plant growthregulator metabolites (Franco and
Coutinho, 1991). Besides agricultural benefits, endophytic actinomycetes can be
used as a source of metabolites for pharmaceutical use (Castillo et al. 2002;
Castillo et al. 2003). In this work, several
isolates recovered were the member of
Streptomyces thermocarboxydus. This
species was revealed as a moderately
thermophilic carboxydotrophic species
from soil (Kim et al. 1998) and has been
isolated from the chromium contaminated
soil, in which its activity was detected as a
Cr(IV)-reducing species (Desjardin et al.
2002). In addition, another strain related
to Nonomuraea sp. was also found to
colonize the tomato plants. To the best of
our knowledge, this is the first report of
the endophytic nature of this genus.
According to Gunnarsson et al. (2003) and
Monciardini & Sosio (2004), Nonomuraea
sp was producer of the antibiotic dalbavancin that had pharmaceutical activity
against gram negative bacteria. Hence, the
current findings may support the previous
claim that endophytic actinomycetes can
be a potential source of the industrially
important compounds (Birber et al. 1998;
Castillo et al. 2003).
The endophytic actinomyecetes population within inside tissues of tomato plants
appears to be much more diverse recovered on HV and TWYE agar media. The
knowledge can lead to selection of the
effective media for isolation of novel and
potentially beneficial endophytes. This
study reveals a novel plant-microbe interaction with implications for biotechnological application of these bacterial
endophytes.

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CONCLUSION
1. The isolates were identified by using
16S ribosomal RNA (rRNA) gene
sequencing and found to belong to a
small group of actinomyecetes genera
including Streptomyces, Microbispora,
and Nonomurae sp. which is first time
found as endophyte.
2. On the basis of isolation media used,
HV agar containing vitamin B and
humic acid as the sole carbon and
nitrogen was more efficient to other
currently used media, including Tap
Water Yeast Extract and Casamino
acid media.

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