Characterization and Antagonistic Activities of Metabolite Produced by Pseudomonas Aeruginosa

Journal of Applied Sciences Research 5(4): 392-403, 2009
© 2009, INSInet Publication
Characterization and Antagonistic Activities of Metabolite Produced by
Pseudomonas Aeruginosa Sha8
Hassanein, W.A., Awny, N.M., El-Mougith, A.A. and Salah El-Dien, S.H.
Department of Botany, Faculty of Science, Zagazig University, Egypt.
Abstract: The antagonistic activities of Bacillus firmus and Pseudomonas aeruginosa which were isolated
from rhizosphere field of Triticum sativum cultivated in El-Sharkia governorate, Egypt; were studied
against 13 fungal isolates. The optimum conditions for growth and maximum production of antifungal
activity were detected against Aspergillus niger, Helminthosporium sp. and Fusarium oxysporium. Only
P. aeruginosa was chosen for more investigation as antifungal agent. King's B medium was used for
production of the antifungal metabolite which extracted by hexane. The extracted metabolite appeared as
one spot on TLC and was characterized by using infra red spectrum (IR), nuclear magnetic resonance
(NMR), mass spectrum (MS) and elemental analysis were detected. The compound was considered as a
derivative of phenazine-1-caboxamide PCN (zag1).The minimum inhibitory concentration of this compound
against A. niger was 23.55µg/ml. The antifungal activity of this compound was remarkable at 100°C for
30 mins. Different morphological and ultrastructure changes induced after treatment of A. niger by
23.55µg/ml PCN (zag1) were recorded by light and transmission electron microscope.
Key words: Pseudomonas aeruginosa, Antagonistic activity and Phenazine compound.
Production of antimicrobial compounds by P.
aeruginosa and its
activity of the increased and
decreased according to the environmental and
nutritional conditions of growth [2 ,1 8 ] .
On the light of these facts this work aimed to
study the biological activities, chemical properties and
identification of the metabolite produced by the isolate
P. aeruginosa Sha8 as well as studying its effects on
A. niger.
INTRODUCTION
Biological control methods aimed to improve the
resistance of the host or favoring microorganisms to
antagonist the pathogen, such as bacteria and fungi[1 3 ].
Control of phytopathogens by biological means was
environmentally advantageous in comparison to
chemical control methods which had many risks on
human health and environment[2 7 ] .
Variable compounds with antifungal and
antibacterial nature were produced by Pseudomonas
spp. and many soil microorganisms such as antibiotics,
iron chelating siderophores, cyanide [1 5 ,3 4 ] and enzymes
such as gluconase, cellulytic and chitinolytic
enzymes[3 0 ] . The activity of these compounds were
reported among antifungal mechanisms by which
Pseudomonas strains inhibited the fungal growth
through damaging of cell walls [1 2 ,3 0 ].
Pseudomonas aeruginosa produced several
metabolites which were active against many pathogenic
fungi and bacteria such as phenazine compound and its
derivatives. There were more than 80 heterotrocyclic
nitrogen – containing natural products of phenazines
synthesized by florescent Pseudomonas spp. [3 ] .
Phenazine-1-carboxamide (PCN) was a derivative which
achieved a broad-spectrum antifungal compound and
consequently act as an effective biological control
agent. It was used as a good antifungal compound
against plant pathogenic fungi as F. oxysporium FOVS
which causes cotton wilt, Rhizocotonia solani RSRI
which caused sheath blight of rice and Pestalotia theae
PTS which caused leaf spot of tea [17 ] .
M ATERIALS AND M ETHODS
Isolation of Bacteria and Fungi: The tested bacteria
were isolated from four different cultivated soil
samples. All isolates were streaked and maintained on
nutrient agar medium [7 ] . On the other hand, the tested
fungi were isolated from infected plants as onion,
tomato and potato. The isolated fungi were streaked
and maintained on Czapek's-Dox agar medium [3 1 ].
Initial Screening for Determination the Antifungal
Activity of the Isolated Bacteria Against Tested
Fungi: The potentialities of the bacteria isolated from
soil samples to produce antifungal compounds were
studied using different assay techniques (filter paper,
agar disc and agar well diffusion). The antagonistic
activity was detected by the appearance of clear
inhibition zones between the fungal and bacterial
growth.
Corresponding Author: Hassanein, W.A., Department of Botany, Faculty of Science, Zagazig University, Egypt.
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J. App. Sci. Res., 5(4): 392-403, 2009
pre-coated 20×20 cm MERCK as described by ElMougith et al [1 1] . The color and Rf values of the spots
were determined.
Identification of the Bacterial and Fungal Isolates:
The most active bacterial isolates were identified
according to Bergey's Manual of Determinative
Bacteriology[1 4 ] while, the isolated pathogenic fungi
were identified according to Moubasher [26 ] .
Identification and Characterization of the Antifungal
Compound: Structure of the purified antifungal
metabolite was established by using the infrared
spectrum (IR), nuclear magnetic resonance (NMR),
mass spectrum (MS) and elemental analysis (carbon,
hydrogen, oxygen and nitrogen). These analysis were
carried out in M icro-Analytical Center, Cairo
University, Egypt).
The minimum inhibitory concentrations (MIC) of
the extracted metabolite produced by P. aeruginosa
Sha8 was determined according to Low et al[2 2 ] against
A. niger. Percent in fungal dry weight inhibition was
calculated. Thermal stability of the purified bacterial
metabolite was determined at 100°C for different
periods against A. niger.
Factors Affecting the Growth and Antifungal
Activities of Both B. Firm us Sha4 and P. Aeruginosa
Sha8: The optimum growth and antifungal activities of
both B. firmus Sha4 and P. aeruginosa Sha8 were
studied at different environmental and nutritional
conditions. The nutrient as well as king's B [1 6 ] broth
media were prepared for cultivation of B. firmus Sha4
and P. aeruginosa Sha8, respectively. Each flask was
inoculated with 1ml of tested bacterial suspension (24
hour). Both bacterial isolates were examined at
different incubation periods (6, 18, 24, 48 and 72
hours), different incubation temperature (15, 25, 35 and
45°C ), different pH values (5.0, 5.6, 6.5, 7.0,7.5 and
8. 0 ), equimolecular amount of different carbon
sources (glucose, galactose, sucrose, starch, fructose,
lactose, mannitol and maltose) and equimolecular
amount different nitrogen sources (NaNO 3 , KNO 3,
NH 4 Cl, casein, urea, gelatin and (NH 4) 2SO 4 under
shaking condition at 185 rpm).
The bacterial growth at each treatment
was
measured at 600 nm by using spectrophotometer
apparatus. All treatments were replicated twice and the
antifungal activity was assayed by using well diffusion
method[24 ] and expressed by measuring the diameters of
inhibition zones (mm).
Electron M icroscopy: Effect of extracted metabolite
on ultra structure of A. niger was examined using light
m icroscope as well as transmission electron
microscopic (Joel-JEM-100 CX, Center of Electron
Microscope -Zagazig University, Egypt) as described
by Lorian [2 0 ].
Statistical Analysis: The obtained data were
statistically analyzed to determine the standard
deviation and differences between means at 5%
probability using T test analysis as described by
Duncan[9 ] . Brivariate correlation matrix of the obtained
data done by using S.P.S.S. software program (Ver,8)
as described by Dytham [1 0 ] .
Production of Antifungal Compound by P.
Aeruginosa Sha8: King's B broth medium was used
for the production of the active metabolite by P.
aeruginosa Sha8. A batch fermentation was performed
in 500 ml shaking Erlenmeyer conical flasks containing
200 ml of the growth medium which inoculated by 10
ml of bacterial suspension (48 hours) at pH 7±0.2 at
30°C and incubated for 48 hours for total metabolite
production.
RESULTS AND DISCUSSION
More efforts had been done for searching more
environmentally friendly methods alternatives to
chemical fungicides through using more useful
microorganisms which can produce some antimicrobial
agents[5,6] . It was found that, biocontrol using
antagonistic bacteria had been considered as alternative
strategy to agrochemicals that were harmful to human
health and environment[1 8 ] .
In this research 60 bacterial isolates were selected,
isolated and purified from the four different cultivated
soil samples.W hile, 13 pathogenic fungal strains were
isolated from different infected plants.
The initial screening of the antifungal activity of
60 bacterial isolates against 13 fungal strains was
carried out by using three techniques; filter paper, agar
disc and well diffusion. 23 bacterial isolates were the
most potent against the 13 tested fungi. The most
active two bacterial isolates were selected for further
Extraction of the Antifungal Compound: At the end
of incubation period, the culture of P. aeruginosa Sha8
was centrifuged at 5000 g for 20 minutes twice in
order to remove the bacterial cells. One liter of
bacterial cell free supernatant was extracted by hexane
(1: 2)[2 8 ] . The extract was examined against the tested
fungi and Saccharomyces cervisiae, Candida albicans
by using filter paper disc.
Thin Layer Chromatography of the Extracted
Antifungal Compound: The purity of the extracted
metabolite compound was assured by using thin layer
chromatography (TLC) aluminum sheets silica gel 60
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J. App. Sci. Res., 5(4): 392-403, 2009
study. These two bacterial isolates were identified as B.
firmus Sha4 and P. aeruginosa Sha8 according to Holt
et al[1 4 ] . The pathogenic fungi were identified as
A.niger, A. flavus, A. fumigatus, A. oryzae, P. citrinum
and P. purpurogenum, Fusarium sp.,F. roseum, F.
oxysporium,
Alternaria sp., Cladosporium sp.,
Helminthosporium sp. and R. solan according to
Moubasher [2 6 ] . In this relation Kumar et al[1 7 ] stated that
P. aeruginosa PuPa3 was an
effective biological
control agent against Fusarium oxysporium which
infected the banana plant. Also, Trivedi et al[3 3 ] proved
the antagonistic activity of Pseudomonas spp. against
two phytopathogenic fungi A. alternata and F.
oxysporium.
Antifungal activity of microorganisms may be due
to production of lytic enzymes, that dergreding the
fu n g a l c e ll w all [ 3 0 ] , a n tib io tic s, F e -c he la ting
siderophores, ammonia and cyanide [2 1 ,3 6 ].
Concerning, the effect of certain environmental
conditions and nutritional requirements on the
antifungal activity of B. firmus Sha4 and P.aeruginosa
Sha8.
It was found that, the maximum growth and
antifungal activity of B. firmus Sha4 illustrated in fig.1
(a, b and c) were obtained after incubation for 24 hr at
30 ° C in nutrient broth adjusted at 7.0 pH these results
were agreement with Bernal et al[2 ] . Also, fig1. (d,e)
illustrated that, the maximum activity of this organism
was achieved when the used medium
was
supplemented with 10 g/l of glucose (as carbon source)
and without addition of any nitrogen source (control).
These results were in agreement with that recorded by
Milner et al[2 5 ] . Similarly, the maximum growth and
antifungal activity of P. aeruginosa Sha8 illustrated in
fig.2 (a, b and c) were obtained, after incubation for 48
hr at 30 ° C in king's B medium which adjusted at 7.0
pH without supplementation of any carbon or nitrogen
source (control) as in fig.2 (d, e). These obtained
results were in agreement with that obtained by
Shaukate and Siddique [2 8 ] . Generally, different factors
affecting the biological activity of microorganisms
depended mainly on the metabolitic pathways as well
as enzymatic system of these organisms [8 ].
Consequently, through this research, P. aeruginosa
Sha8 was chosen for further studies.The Selection of
this organism was not only for its highly antifungal
activity but also this bacteria possessed many traits that
make them also well studied as biocontrol[3 7 ] including
grow rapidly, colonized and multiplied in rhizospere,
aggressively with other microorganisms and adapted
to environmental stress[3 6 ] .
Nine organic solvent were used for extraction of
the metabolite produced by P. aeuginosa sha8 Nbutanol, chloroform, ethyl acetate, benzene, diethyl
ether, petroleum ether, hexane and mix chloroform,
methanol were used in percent of 1:1 (v/v) except
hexane was 2:1(v/v) as illustrated in table (1). The
extracts were examined against the tested fungi. The
results indicated that, diameter of inhibition zones were
23.0 mm, 20.0 mm and 21.0 mm for A. niger,
Heminthosporium sp. and F. oxysporium respectively,
while C. albicans and S. cerevisiae strains were not
affected by the extracted metabolite in case of
hexane.The other tested organic solvent for extraction
were not able to extract the desirable metabolite except
ethyle acetate which showed inhibition zones against
Heminthosporium sp. and F. oxysporium as appeared in
table (1).
Purity of the extracted metabolite was achieved by
TLC analysis under U.V rays at 365 and 254 nm after
spraying with 20% sulphoric acid and heated in oven
at 100ºC for 10 minutes. The results represented in
fig. 3,4 indicated that, extracted metabolite had one
spot with R f 0.5 ± 0.02 . The obtained results was found
similar to that detected by Kumar et al[1 7 ] .
It was found that, the purified metabolite was a
viscous greenish yellow substance miscible in water
and dissolved in hexane. This metabolite was
characterized by different physic-chemical studies as
infrared spectrum (fig.3), nuclear magnetic resonance
(fig.4), mass spectrum (fig.5) and elemental analysis.
The results indicated that, C 1 4H 1 3N 3O 2 was the
suggested empirical formula for this metabolite the
metabolite component which considered as derivative
of phenazine-1-carboxamide (PCN) which have the
empirical formula C 13H 1 0 N 3 O.
The summarized results based on the IR,H 1 NMR,
M S spectrum and elemental analysis indicated that, this
metabolite had a broad- spectrum antifungal propriety,
it was identified as a derivative of phenazine-1carboxamide acid (PCN zag1). Earlier studies had
reported the production of phenazine-1-carboxamide
(PCN) from Pseudomonas strains as P. chlororaphis
PCL1391[4 ] , P. aeruginosa PA01 [2 3 ] and P. aeruginosa
PUPa3 [1 8 ] . Some strains of fluorescent Pseudomonas as
P. fluorescens 2-27 and P. aureofaciens 30-84
produced phenazine derivatives as phenazine-1carboxylic acid (PCA) which considered yellow fine
needles and it's empirical formula was C 1 3H 9N 2O 2.
Some pathogenic fungal strains as Gaeumannomyas
graminis var. tritici which caused root disease of wheat
was suppressed by this compound [3 2 ] . It was
documented that the biocontrol activity of PCN was
found to be 10 times higher than PCA in neutral pH [4 ].
Through this research, different properties of the
PCN (zag1) were studied. The minimum inhibitory
concentration of this compound against A. niger was
23.55µg/ml.Concerning to the thermal stability of PCN
(zag1), data were represented in fig. (6) it indicated
that, the antifungal activity of this compound was
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J. App. Sci. Res., 5(4): 392-403, 2009
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J. App. Sci. Res., 5(4): 392-403, 2009
Fig. 1: Antifungal activities of B. firmus Sha4 against different fungal species under different environmental and
nutritional conditions.
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J. App. Sci. Res., 5(4): 392-403, 2009
Fig. 2: Antifungal activities of P. aeruginosa Sha8 against different fungal species under different environmental
and nutritional conditions.
Table 1: Efficiency of different organic solvents to extract the antifungal m etabolite com pound produced by P. aeruginosa Sha.8.
Tested fungi
D iam eter of inhibition zone ( m m )
O rganic solvents
---------------------------------------------------------------------------------------------------------------A. niger
H elm intho-sprium sp
F.oxysporium
C. albicans
S. cerevisiae
N -butanol
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Chloroform
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Ethyleactate
15.0
14.0
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Benzene
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------D iethyleether
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Petrolum ether
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Toluene
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------H exane
23.0
20.0
21.0
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------M ixture 9:1(v/v) chloroform +m ethanol
Control; solvent without extract.
(-) : no inhibition zone.
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J. App. Sci. Res., 5(4): 392-403, 2009
Fig.3: IR spectrum of extracted metabolite.
Fig. 4: H1 NMR of extracted metabolite.
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J. App. Sci. Res., 5(4): 392-403, 2009
Fig. 5: Mass spectrum of extracted metabolite.
Fig. 6: The thermal stability of phenazine -1-carboxamide zag1 produced by P. aeruginosa Sha8 against A. niger.
remarkable at 100 " C for 30 min. Also, 23.55 mg/ml
was the minimum inhibitory concentration for the
extracted metabolite against A. niger. This inhibitory
through the effect of this compound on the metabolic
fungal path ways was previously proposed by Low
et al[2 1 ].
It is appeared that, by increasing the time of heat
exposure of (11.77 ìg/ ml) dose from PCN (zag1) at
100°C decreased its activity and consequently,
increased the fungal growth. Also, it was found that,
the metabolite was activity remarkable for 30 min at
100 °C.In this relation W ang et al[3 5 ] denoted that, the
culture of P. aeruginosa K-187 was thermal stable up
to100° C and maintained around 90% of its activity
even a high temperature for 30 min. Lossing the
antifungal activity of the compounds after high
temperature treatment for long time may be related to
chemical configuration or and decomposition of this
component due to the effect of temperature on its
chemical bonds, and consequently loss its biological
activity.
To study the effect of this compound on A. niger,
(11.77 ìg/ml) dose of phenazine-1-carboxamide (zag1)
was used when aseptically added to the growth
medium of A. niger; it induced a morphological and
abnormalities during germination stages of tested A.
niger by using light microscope. It was appeared that
the presence of phenazine -1-carboxamide zag1 in the
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J. App. Sci. Res., 5(4): 392-403, 2009
fungal growth medium induced numerous harmful
effects which leaded to the growth reduction. These
harmful effects were clearly appeared on studying the
ultra structure of the treated and non treated germinated
spores. The non treated spores germinate regularly in
suitable time where they gave normal germ tube as
illustrated in plate (²-A) which leaded to formation of
normal and branched mycelia.
The germination of treated spores with phenazine
-1-carboxamide zag1 was delayed and they changed to
amorphous structure which germinate from numerous
position plate (² -B) and also gave more than one
anomalous germ tube which lost their polarity of
growth and with abnormal apex then these spores
become highly vacuolated. Other spores failed to
germinate as illustrated in plate (²-B).
The presence of phenazine-1-carboxamide zag1,
delayed the germination. The mature treated mycelia
have delicate and ruptured cell wall, especially the
external layer of cell wall. Also, the treated mycelia
became wider and vacuoles were formed, these
vacuoles may contained electron dense bodies plate (²C). These treated mycelia go on lysis as appeared in
plate (² -D).
Plate I: Photomicrographs showing the effect of
Phenazine-1-carboxamide (zag1) on the
different structure of A. niger as appeared in
semi- thin sections (x 400).
A
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J. App. Sci. Res., 5(4): 392-403, 2009
D
B
Plate ²²: Electron microscope showing the effect of
phenazine-1-carboxamide (zag1) on the ultra
structure of A. niger C.W .,cell wall; P.M.,
p lasm a membrane; T .Sep ., transfer
s e p tu m ;M ., m ito c ho nd ria ;N .,nuc le u s ;
Nu.,nucleolus; va., Vacuole; E.B., electron
d e nse b o d y;G .T ., ge rm tub e ; cyto .,
Cytoplasm; R., ribosome and Ly.C., lysed
cell.
In this connection, several apparent mechanisms
for the antifungal compounds involved in the
antagonism of pathogenic fungi had been previously
proposed. They includied interference with spore
germination or germ tube elongation [2 9 ], inhibition
through abnormal hyphal swelling [1 ] and lysis of the
hyphal lip [19 ] . Also W ang et al[3 5 ] concluded that
chemical compounds affected the fungal growth
through the interference in metabolic pathways and
induced ultrastructure variations.
One particularly attractive characteristic that P.
aeruginosa enhanced disease protection and improve
consistency of a biological control agent where, the
effective utilization of such agent promoted the
economic yield of the cultivated plant. Development of
such P.aeruginosa as a biocontrol agent was an
environmentally benign alternative to current disease
control strategy[3 5 ].
C
ACKNOW LEDGEM ENTS
The authors gratefully acknowledgement
financial support of the Zagazig university.
401
the
J. App. Sci. Res., 5(4): 392-403, 2009
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