173

NSave Nature to Survive

ISSN: 0974 - 0376

: Special issue, Vol. 1;

Paper presented in 3rd International Conference onClimate Change, Forest Resource and Environment

(ICCFRE, 2011)December 09 - 11, 2011, Thiruvananthapuram,

organized byDepartment of Environmental Sciences,

University of Keralain association with

National Environmentalists Association, Indiawww.neaindia.org

QUARTERLY

www.theecoscan.in

KEYWORDS

I. Abdul Kafur and Anisa B. Khan

Medicinal plants

Endophytic Actinomycetes

Antibacterial activity

173 - 177

ISOLATION AND SCREENING OF ENDOPHYTIC ACTINOMYCETES

AGAINST HUMAN BACTERIAL PATHOGENS

174

I. ABDUL KAFUR AND ANISA B. KHAN*

Department of Ecology and Environmental Sciences,

Pondicherry University, Puducherry - 605 014, INDIA

E-mail: anisabasheer@gmail.com

INTRODUCTION

Within the last decade, it has become increasingly evident that there is enormous

microbiological diversity residing within the tissues of plants (Idris et al., 2004;

Reiter and Sessitsch, 2006; Smith et al., 2008). The ecological role of the endophyte

to the plant remains uncertain, although the presence of endophytic

microorganisms in the plant host in most cases is beneficial for the plant. Secondary

metabolites produced by endophytes provide a variety of fitness enhancements

such as increased resistance to herbivore, parasitism, drought, besides promoters

of growth. The list of novel actinomycetes and products derived from poorly

explored areas of the world, stresses the importance of investigating new habitats

(Nolan and Cross, 1988). Selective isolation of actinomycetes from plants is

important for obtaining new strains and to study their ecology. The advent of the

development of drug resistance in pathogenic bacterial, fungal infections and

certain cancers has prompted a search for more and better agents (Strobel and

Daisy, 2003). It now appears that endophytes are relatively untapped sources of

novel natural products and streptomycetes also exist as endophytes within the

living tissues of higher plants and serve as sources of novel bioactive products

(Castillo et al., 2007; Zin et al., 2007; Ryan et al., 2008). Igarashi et al. (2002)

isolated 398 actinomycete strains from leaves, stems and roots of cultivated and

wild plants. About 10-20% of the n-butanol extracts of their fermentation broths

showed antagonistic activity against phytopathogenic fungi and bacteria. Igarashi

et al. (2000) found that such an effect was dependent on a protective compound

named fistupyrone. Their results suggest that a wide range of endophytic

actinomycetes have a potential to produce antimicrobial compounds. Sasaki et

al. (2001a,b) identified several new bioactive compounds produced by

actinomycetes isolated from live plants. Two new novobiocin analogs produced

by Streptomyces sp. collected off Aucuba japonica (Sasaki et al., 2001a) and

cedarmycins by Streptomyces sp. collected from Cryptomeria japonica (Sasaki et

al., 2001b) were determined to be antimicrobial metabolites. A new

naphthoquinone antibiotic, alnumycin, was also reported in Streptomyces sp.

isolated from root nodules of Alnus glutinosa collected in Germany (Bieber et al.,

1998). Shimizu et al. (2004) proved that Streptomyces galbus strain R-5 produced

actinomycin X2 and fungichromin that could account for the in vitro anti-bacterial

and anti-fungal activities.

A search for specific endophytes that may produce antibiotics can have its origin

in ethnobotany, which utilizes the medicinal lore of native people (Strobel, 2003).

On the other hand, Streptomyces sp. NRRL30562 obtained from snakevine plant

produced novel peptide antibiotics, designated as munumbicins A-D (Castillo et

al., 2002). These antibiotics possessed a wide-spectrum activity against many

human and phytopathogenic fungi and bacteria. Castillo et al., 2003 found that

Streptomyces sp. NRRL30566, which was isolated from a fern-leaved grevillea

(Grevillea pteridifolia) tree growing in the northern territory of Australia, produced

novel wide-spectrum antibiotics named kakadumycins that were chemically related

to echinomycin.*Corresponding author

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Endophytic actinomycetes were isolated from

surface-sterilized leaves of four medicinal

plants, Catharanthes roseus, Azadirachta

indica, Citrus limon and Mentha arvensis. A

total of 30 endophytic actinomycete isolates

were obtained. The highest number of

actinomycetes were recovered from C. roseus

leaves representing 66.6% of the total isolates.

Submerged culture broth of all cultures were

assayed for antibacterial activity against

Bacillus subtilis, Staphylococcus aureus,

Pseudomonas aeruginosa and Proteus vulgaris

by agar well diffusion method. Among the 30

isolates, 22 (73%) isolates showed activity

against test microorganisms while 8 (27%)

isolates were found to be non active in primary

screening. Results of the present study indicate

the richness of bioactive microbial diversity in

medicinal plants and their screening for

antibacterial activity should be intensified for

identification and application as novel source

of bioactive compounds.

ABSTRACT

175

However, the endophytic community of many of the medicinal

plants has not yet been investigated. The present study

describes the isolation of actinomycetes from the internal

tissues of some medicinal plants of Puducherry region and

evaluation of their metabolites for antibacterial activity.

MATERIALS AND METHODS

Sample collection

Location and collection of plant leaf sample: Leaf samples

were collected from randomly selected healthy wild and

cultivated plants within the university campus, at Puducherry,

India. Samples were placed in clean plastic bags, brought to

the laboratory and used for isolation purpose. The following

plants were used as source for isolation of endophytic

actinomycetes.

Catharanthes roseus (L.) G.Don (Apocynaceae), Citrus limon

(L.) Burm.f. (Rutaceae), Mentha arvensis L. (Lamiaceae),

Azadirachta indica (Meliaceae).

Isolation medium

Endophytic actinomycetes were isolated on Starch Casein Agar

(SCA) (Küster and Williams, 1964) which contained g/L.

(soluble starch 10g; KNO3 2g; NaCl 2g; K

2HPO

4 2g; MgSO

4.

7H2O 0.05g; CaCO

3 0.02g; FeSO

4. 7H

2O 0.01g; Casein 0.3g;

Agar 18g; Distilled water 1000 mL; pH 7). The isolates were

maintained on Potato Dextrose Agar (PDA) (Potato 200g;

Dextrose 20g; Agar 18g; Distilled water 1000 mL and pH 7).

For fermentation studies, Yeast extract- Malt extract broth (ISP2)

(Shirling and Gottlieb, 1966) (Yeast extract 4 g; Malt extract 10

g; Dextrose 4 g Distilled water 1000 mL and pH 7) were used.

Test microorganisms

The following test microorganisms procured from Microbial

Type Culture Collections and Gene bank (MTCC) at

Chandigarh, India, were used during the investigation: Bacillus

subtilis (MTCC 441), Staphylococcus aureus (MTCC 96),

Pseudomonas aeruginosa (MTCC 424), Proteus vulgaris

(MTCC 744). Bacterial cultures were maintained on nutrient

agar (NA) slants in a refrigerator at 4ºC for routine use.

Isolation of endophytic actinomycetes from leaves

The leaf samples were washed in running tap water for 10

minutes and cut into small bits (1.0 x 0.5 cm). The leaf segments

were surface sterilized following the method of Taechowisan

and Lumyong, (2003). Leaf bits were rinsed in 0.1% Tween 20

for a few seconds, then transferred to clean conical flask and

sterilized by sequential immersion in 70% (v/v) ethanol for 60

seconds, followed by sodium hypochlorite solution (1% w/v,

available chlorine) for 120 seconds and finally in 70% ethanol

(v/v) for 30 seconds and then inoculated on SCA at the rate of

10-15 leaf bits per plate. Nystatin and cycloheximide (50μg/

mL of each) were added to the isolation medium to suppress

fungal growth (Williams and Davies, 1965). All the plates were

incubated under laboratory conditions at 26±2ºC for 15-30

days. Individual colonies with characteristics of Actinomycete

morphology were isolated and pure cultures of the respective

isolates were obtained by repeated streaking on SCA plates.

Pure isolates were transferred to freshly prepared PDA slants

and preserved at 4ºC. These isolates were evaluated for their

antimicrobial activity.

Test for effectiveness of surface sterilization

To validate the sterilization procedure, ten surface sterilized

leaf segments were soaked in 10 mL sterile water and stirred

for 3min. An aliquot of 0.3 mL suspensions were then

inoculated on nutrient agar plates, sealed with parafilm and

incubated at 26±2ºC for 14 days and checked for

development of any bacterial colony.

Antibacterial activity of endophytic actinomycetes isolates

against human bacterial pathogens

Fifty mL ISP2 was inoculated with 0.5 mL spore suspension of

the selected isolates in a flask and incubated under static

conditions at room temperature (28±2ºC) for 15 days. The

contents of the flasks were filtered separately through a cotton

pad to exclude spores and mycelial mass. The clear filtrate

was centrifuged at 5000 rpm for 10 minutes and the clear

supernatant was transferred to sterile conical flaks and stored

in freezer for further processing.

A total of 30 aerobic well sporulating isolates were screened

for inhibitory activity on the test bacteria by in vitro well

diffusion assay techniques and these tests were conducted on

PDA in petri dishes.

Antimicrobial activity of the culture broth was tested by agar

well method against four species of bacteria namely B. subtilis,

S. aureus, P. aeruginosa, P. vulgaris. Bacteria grown on nutrient

broth (pH 7) for 24 h were surface inoculated onto sterile PDA

agar plates using sterile cotton swabs. Wells were made in

each plate using sterile 6 mm diameter cork borer. Crude

supernatant of each actinomycete strain (50μL) was added

separately in to each well and incubated at room temperature

for 24-48 h at the end of which bacterial growth was observed

and the zone of inhibition was measured and recorded. The

intensity of inhibition was noted as follows: ++ = 11-19

mm, + = 2-10 mm; ± = < 1 mm; - = 0 mm.

RESULTS AND DISCUSSION

Isolation of Actinomycetes

Thirty actinomycete isolates (Table 1) obtained in pure form

from the four medicinal plants were evaluated for their

antibacterial activities. Pure cultures were maintained on PDA

medium. Development of the endophytic actinomycetes on

the leaf of host plant showed hyphal growth which

subsequently grew out onto the surface of the SC agar (Fig.

1a). These actinomycetes were examined by direct scanning

electron microscopy (Fig. 1b).

Screening for antibacterial activity

In vitro screening for antibacterial activity showed that, out of

30 isolates, 22 (73%) isolates showed strong to mild activity

against the test microorganisms while 8 (27%) isolates were

found to be inactive in primary screening in the fermentation

broth.

The intensity of inhibition (measured in terms of diameter of

inhibition zone) against the test microorganisms is presented

Table 2. Thirteen (43.33%) isolates inhibited both gram positive

and gram negative bacteria. Among them 6 (20%) isolates

inhibited only gram positive bacteria and 3 (10%) isolates

inhibited only gram negative bacteria. No isolate inhibited all

ENDOPHYTIC ACTINOMYCETES

176

I. ABDUL KAFUR AND ANISA B. KHAN

the test bacteria. Out of the four bacteria tested B. subtilis was

more sensitive and was inhibited by 15 isolates followed by P.

vulgaris (15 isolates), S. aureus (13 isolates) and P. aeruginosa

(6 isolates). Antibacterial screening indicated that nine isolates

inhibited a maximum of three test bacteria; nine isolates

inhibited two test bacteria and four isolates inhibited one test

bacteria.

The present finding highlights the importance of extensive

investigation of the precious biowealth for extracting novel

antimicrobial agents out of the actinomycetes, from the

unexplored area in this field. The emergence and dissemination

of antibacterial resistance is well documented as a serious

problem worldwide (Cohen, 2000), showing that the potencies

of prevalent antibiotics are decreasing steadily leading to

reduced effectiveness of drugs. This situation compounds the

need for the investigation of new, safe and effective

antimicrobials for not only replacement of invalidated

antimicrobials but also for use in antibiotic rotation programs

(Gerding et al., 1991). However production of metabolites

useful in pharmaceutical industry is widespread, mostly

among endophytic fungi. In this study, endophytic

actinomycetes associated with pharmaceutical plants which

displayed remarkable antibacterial activities and appear to be

a promising perspective for improved poduction of

pharmaceutical agents. Thus for the biotechnological

production of natural compounds endophytes seem to be the

most interesting alternative and they are likely to play more

significant role in the years to come. Further study on the

bioactive metabolites produced by the best isolates that exhibit

wide spectrum activity is under progress.

ACKNOWLEDGEMENT

Authors thank Pondicherry Federal University for the

infrastructural facilities. One of the authors acknowledges

University Grants Commission for the funds in carrying out

Plant Source Plant part used No. of actinomycete isolates

Catharanthes roseus Leaf 20

Azadirachta indica Leaf 7

Citrus limon Leaf 2

Mentha arvensis Leaf 1

Table 1: Isolation of endophytic actinomycetes from leaves of

medicinal plants

Actinomycetes Bacillus Staphylococcus Proteus Pseudomonas

Isolates subtilis aureus vulgaris aeruginosa

Cr 01 - - - -

Cr 02 - - - -

Cr 03 ++ ++ - -

Cr 04 ++ ++ ++ -

Cr 05 - - - -

Cr 06 - - - -

Cr 07 - - - -

Cr 08 - - - -

Cr 09 - ++ ++ +

Cr 10 ++ - ++ -

Cr 11 ++ - ++ +

Cr 12 ++ ++ ++ -

Cr 13 - - ++ +

Cr 14 - ++ - ++

Cr 15 + - + -

Cr 16 - - - -

Cr 17 - - + ++

Cr 18 ++ - + +

Cr 19 ++ ++ - -

Cr 20 ++ ++ ++ -

Ai 1 + + - -

Ai 2 + - - -

Ai 3 - + + -

Ai 4 - - + -

Ai 5 ++ - - -

Ai 6 - + - -

Ai 7 - - - -

Cl 1 ++ + ++ -

Cl 2 ++ ++ ++ -

Ma ++ ++ ++ -

Cr: Catharanthes roseus, Ai: Azadirachta indica, Cl: Citrus limon, Ma: Mentha arvensis.

++ = 11-19 mm, + = 2-10 mm; ± = < 1 mm; - = 0 mm.

Table 2: Antibacterial activity of endophytic actinomycetes isolated

from four medicinal plant leaves

Figure 1b: Scanning electron micrograph of aerial hyphae of

actinomycetes on leaf bits

Figure 1a: Isolation of foliar endophytic actinomycetes

177

this work.

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