Narasimha Reddy Parine et al.

/International Journal of Chemical and Analytical Science 2010,1(5),94-96

Research Article
ISSN: 0976-1209 Available online through
www.ijcas.info
Antibacterial Efficacy of Secondary Metabolites from Entomopathogenic Fungi Beauveria
bassiana
Narasimha Reddy Parine1 , Akbar Ali Khan Pathan2 , Sarayu B3 , Veeturi Sudha Nishanth2 , Varaprasad Bobbarala4*
1
Centre for Cellular and Molecular Biology, Habsiguda, Hyderabad, India
2
Department of Biotechnology, GIT, GITAM University, Visakhapatnam, India
3
Enviromental Life Sciences, University of Salford, Greater Manchester, UK
4
Vivimed labs Limited, 2nd, 4th Floor, Veeranag towers, Habsiguda, Hyderabad, A.P.

Received on: 21-01-2010; Revised on: 18-03-2010; Accepted on:22-04-2010

ABSTRACT
This study was performed to evaluate the antibacterial activity of crude ethyl acetate extract of the entomopathogenic fungi Beauveria bassiana (ITCC 4688)
against ten aerobic pathogenic bacteria including Bacillus megaterium, Staphylococcus aureus, Escherichia coli, Streptococcus pyogenes, Micrococcus luteus,
Bacillus subtilis, Bacillus sphaericus, Klebsiella aerogenes, Psuedomonas aeruginosa and Chromobacterium violaceum. The acetate extracts of Beauveria
bassiana (ITCC 4688) were shown to possess an inhibiting activity (MIC < or = mg/L) against many of the species tested. Significant antibacterial activity was
found from 500 to 1000 µg/ml against Bacillus megaterium, Bacillus subtilis, Bacillus sphaericus, Escherichia coli; 700 to 1000 µg/ml against Micrococcus luteus,
Pseudomonas aeruginosa and from 900 to 1000 µg /ml against Streptococcus pyogenes, Chromobacterium violaceum. The results suggest differential action of
antibacterial property of crude extract of B. bassiana depending upon the active compounds on the specific bacteria. Hence, the present study gives an idea to test
against more number of organisms and to find out the actual antibacterial compound from B. bassiana.

Keywords: Beauveria bassiana, entomopathogenic fungi, antibacterial activity, MIC

INTRODUCTION

Fungi play a major role in soil ecosystems along with bacteria,
protists, small invertebrates and plants, through complex trophic interac-
tions. Most soil fungi are regarded as saprobes, decomposing organic matter
and contributing to nutrient cycling, while several species form mycorrhizal
associations with plants or are plant pathogens1. Also recognized as prolific
secondary metabolite producers, fungi have provided several bioactive com-
pounds and chemical models currently used as pharmaceuticals, and soils
are traditionally the main source of fungal genetic resources for bioprospection
programs 2.
Fungi produce a wide range of secondary metabolites with high
therapeutic value as antibiotics, cytotoxic substances, insecticides, com-
pounds that promote or inhibit growth, attractor, and repellent etc.3 Second-
ary metabolites produced from fungi vary in production, function and specifity
to a particular fungus 4. Entomopathogenic fungi have been investigated for
use against a broad range of insect pests5, 6, 7, 8. Diverse toxic metabolites
have been described in several fungal biological control agents including
species of Beauveria, Metarhizium, and Paecilomyces 9. Some of these
metabolites have been found to display antibiotic, fungicidal or insecticidal
properties against insect pests and diseases9, 10. B. bassiana is a fungal
contact pathogen, acting as a microbiological insecticide. The conidium ofB.
bassiana adheres to the insect cuticle and infects the insect by mechanical
and enzymatic activity. B. bassiana produces many mycotoxins in insect
*Corresponding author.
Dr. Varaprasad Bobbarala M.Sc., Ph.D.
Head-Natural Products Chemistry
Vivimed labs Limited,
Tel.: + 91-9949129539
E-mail:varaprasadphd@rediffmail.com
hemocoel probably to overcome insect defences11. Among them, the low
molecular weight compounds-Beauvericin, Enniatins, Isarolides and
Bassianolide have been demonstrated to be insecticidal12, 13, 14 . Evidence for
the involvement of fungal toxins in antimicrobial activity preventing second-
ary infections in silkworm from other bacteria has been reported by Kodaira15.
Production of oosporein is commonly observed in some cultures of this
fungus.
In our recent study on endophytic nature of B. bassiana,
we have observed that B. bassiana is protecting the sorghum plants from
insect pests and fungal pathogens like Myrethecium roridum16 . This may be
due to secondary metabolites released by endophytic fungi in plant 16 . Sec-
ondary metabolites of B. bassianaare reported to have antifungal properties
against some fungal pathogens17 . Therefore, in the present study we fo-
cused on the antibacterial activity of B. bassiana secondary metabolites.
MATERIALS AND METHODS
Fungal growth and extraction of crude extract
Beauveria bassiana isolate ITCC 4688 (Indian type culture
collection, IARI, Delhi, India) that showed endophytic nature in sor-
ghum18 and also caused ~80% mortality on larvae of Helicoverpa
armigera in laboratory bioassays16 was selected for the experiment.
Beauveria bassiana cultures grown on Sabouraud’s Dextrose Agar
(SDAY) for 14 days were used. Conidia were harvested by lightly scrap-
ing the fungal surface with a sterile scalpel19 . One ml of aqueous conidial
suspension (107 conidia/ml) was inoculated in 100 ml liquid medium in
250 ml Erlenmeyer flasks 20 . The flasks were incubated on a rotary shaker
(Remi Pvt. Ltd., India) at 180 rpm for 7 days at 25 ± 2 ºC.

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Table 1 : Testing of antifungal activity of B. bassiana against some selected bacterial strains

Test Organism Concentration (µg/ml) †¥ Streptomycin 100 µg/ml Penicillin G 100 µg/ml

100 200 300 500 700 900 1000

Bacillus megaterium – – 4.5±1.2 11.3±1.9 16.3±2 19.6±1.6 21±0.9 – 23.34±1.89
Bacillus subtilis – – 3.1±0.6 10±0 15.9±1.3 21±1.3 24.8±0.6 – 26.4±1.33
Bacillus sphaericus – – 4.3±1.9 12.6±1.6 15.4±2.3 21.6±3 22.8±1.8 – 25.6±1.73
Micrococcus luteus – – – 3.8±2.3 9.3±1.6 16.1±2.3 24.3±1.3 – 27.56±3.33
Staphylococcus aureus – – – – – – – – 22.66±2.3
Streptococcus pyogenes – – – – 5.1±1.3 11±0.3 17.9±1 – 28.36±2.6
Chromobacterium violaceum – – – – 3.2±0.3 6.9±1.3 11.2±0.9 27.1±2.8 –
Escherichia coli – – 4.1±0.3 12.3±1.9 19±2.3 27.6±2.3 31.2±1.9 34.66±1.9 –
Klebsiella aerogenes – – – – – – – 21.33±1.33 –
Pseudomonas aeruginosa – – – 5.6±0.6 9.3±0.6 17.3±0.9 19.6±2.1 23.33±2.66 –

†
Zone of Inhibition values are indicated in mm; Negative control (DMSO) – No activity.
¥
Values represent mean (± SE) of three experiments each set up in triplicate

The fermented broth (3 liters) was treated with ethyl acetate (2
liters) for 2 hours followed by cheesecloth filtration to remove the biom-
ass. The organic extract was separated and dried over anhydrous so-
dium sulfate and concentrated in vacuo to yield a crude yellow solid
(0.3g).
Bacterial Cultures
Ten bacterial organisms’ Bacillus megaterium (MTCC 453),
Staphylococcus aureus (MTCC 7202), Escherichia coli (MTCC 581),
Streptococcus pyogenes (MTCC 442), Micrococcus luteus (MTCC
2847), Bacillus subtilis (MTCC 441), Bacillus sphaericus (MTCC 511),
Klebsiella aerogenes (MTCC 39), Psuedomonas aeruginosa (MTCC
741) and Chromobacterium violaceum (MTCC 2656) were obtained
from the Institute of Microbial Technology (IMTECH), Chandigarh,
India. Cultures were maintained on nutrient agar slants and were sub-
cultured in petri dishes prior to testing.
Antibacterial studies:
Antibacterial activity was examined according to the method
described previously by Ansari et al., 8 . The anti-microbial activity of
crude ethyl acetate extract of B. bassiana was determined using a paper
disc diffusion assay. In this test each disc (0.5 cm dia; Hi-Media, India)
was treated with different concentrations (100 - 1000 µg/ml) of B.
bassiana crude extract. Three treated and one control (solvent only)
discs were placed perpendicular to each other on freshly inoculated
bacterial cultures in a 12-cm dia. Petri dish with nutrient agar medium.
To inoculate with bacteria, 100 µl of 1×10 cfu/ml were spread
9
over the nutrient agar plate and incubated at 33 ± 1°C resulting in a
bacterial lawn within 48h. The presence of zones of inhibition in the
bacterial lawn was interpreted as a positive activity. Penicillin G (100 µg/
ml) and streptomycin (100 µg/ml) were used as positive controls and
DMSO was used as negative control. After the incubation radial growth
on the media containing the sample was divided by the growth of con-
trol to provide the percentages of growth. Minimum inhibitory concen-
trations (MICs) which completely inhibited bacterial growth were also
indicated. The diameter of the minimum zone of inhibition was mea-
sured. All assays were set up in triplicate. The assays were repeated
three times.
DATA ANALYSIS
The values in all experiments were arcsine percent square root
transformed, and the mean ± SE in each treatment was back-transformed
to normalize the data21 .
RESULTS AND DISCUSSION
Fungi are well known to show antibacterial, antifungal, larvi-
cidal, molluscicidal, antioxidant and free-radical scavenging activities 4 .
In the present study, different concentrations of crude ethyl acetate
extract of B. bassiana were tested against ten bacterial strains. It is
found that there was no significant antibacterial activity exhibited by
any concentrations of B. bassiana crude extract on Staphylococcus
aureus and Klebsiella aerogenes (Table.1). There was significant anti-
bacterial activities found from 500 to 1000 µg/ml against Bacillus
megaterium, Bacillus subtilis, Bacillus sphaericus, Escherichia coli;
700 to 1000 µg/ml against Micrococcus luteus, Pseudomonas aeruginosa
and from 900 to 1000 µg /ml against Streptococcus pyogenes, Chromo-
bacterium violaceum (Table.1). However, these antibacterial activities
against these bacteria were shown to be equal or less active when
compared to the controls Streptomycin (100 µg/ml) and Penicillin G.
This differential action of antibacterial property of crude extract of B.
bassiana may be depending upon the active compounds on the spe-
cific bacteria. Hence, the present study gives an idea to test more num-
ber of organisms and to find out the actual antibacterial compound from
B. bassiana.
ACKNOWLEDGEMENT
Veeturi Sudha Nishanth is thankful to the Department of Bio-
technology, GIT, GITAM, University, Visakhapatnam for providing nec-
essary facilities to carrying out this work.
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Source of support: Nil, Conflict of interest: None Declared

International Journal of Chemical and Analytical Science Vol.1.Issue 5.May 2010 94-96