3. isolation of endophytic actinomycetes using surface sterilization method and selectively...

Jurnal Agrisistem, Juni 2009, Vol. 5 No. 1 ISSN 1858-4330

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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 plant-

associated bacteria is important to under-

stand 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 phyto-

patogens. Among the bacterial endo-

phytes, actinomycetes have received con-

siderably attention due to their long time

prominence as the main source of wide

spectrum of antibiotics and other bene-

ficial 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 actinomy-

cetes have been successfully isolated from

a wide variety of plants, such as rhodo-

dendron (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-

gens and therefore may be better accli-

matized 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 bio-

active compounds. Therefore, this study

was designed to develop an effective me-

thod on isolation of endophytic actino-

mycetes 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 ste-

rilization procedure. The plant segments

were immersed in 99% ethanol for 60

seconds, followed by a 12 minute im-

merse in 6% NaOCl and a 60 second wash

in 99% ethanol. The surface-sterilized

plant segments were rinsed in sterile


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Reverse Osmosis (RO) water and asep-

tically sectioned into 1 cm long fragments.

Finally, the plant fragments were dis-

tributed 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 subcul-

tured media: a half strength Potato

Dextrose Agar (PDA) and Mannitol Soya

flour agar (MS) plates. Actinomycetes

growing on the media were purified and

identified.

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-

doxine-HCl, inositol, Ca-pantothenate, p-

aminobenzoic 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.

The isolates are most prevalent from roots

(56.66%), leaves (26.66%) and less from

stems (16.66%).

36.11%

58.33%

5.50%

0

0.1

0.2

0.3

0.4

0.5

0.6

TWYE HV YECD

The percentage of effectiveness of each

isolation medium

Figure 1. Percentage of effectiveness of isolation media


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Table 1 illustrates the number of actino-

mycete 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

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 17 56.66

Stems 5 16.66

Leaves 8 26.66

All isolates were first identified morpho-

logically. 36 different isolates based on

morphology characterization were then

subjected to 16S ribosomal gene sequen-

cing (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 pla-

ced in the genus Microbispora according

to the closest match sequences found in


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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 ob-

tained can be considered to be true endo-

phytes. 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. There-

fore, it is acknowledged that the actino-

mycetes 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 pro-

duced higher numbers of actinomycete

colonies than the two other media. The

superiority of HV agar is related to the

special activity of actinomycetes in natu-

ral environment and to the property of

humic acid which activate actinomycetes

spores upon germination (Hayakawa and

Nonomura 1987). On the HV agar, Acti-

nomycetes colonies developed well and

formed spores or sporangia quite abun-

dantly from the aerial or substrate my-

celia. Another medium in which actino-

mycete colonies appear relatively abun-

dant was TWYE agar. This low nutrient

medium can retarded the growth of fungal

and eubacteria sufficiently to allow incu-

bation of plates for 7 to 14 days, by which

time actinomycete colonies grew and

sporulated (Figure 2). The present of the

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-


27

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 growth-

regulator metabolites (Franco and

Coutinho, 1991). Besides agricultural be-

nefits, endophytic actinomycetes can be

used as a source of metabolites for phar-

maceutical 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 dal-

bavancin 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 popula-

tion within inside tissues of tomato plants

appears to be much more diverse reco-

vered 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 inter-

action with implications for biotech-

nological application of these bacterial

endophytes.

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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