Mycoflora associated with Halfa-bar leaves and stems (Cymbopogon schoenanthus L. Sprenge), in vitro the antimicrobial activity of the plant leaves and stems secondary metabolites

Abstract

Cymbopogon schoenanthus L. Sprengel, Family Poaceae, locally known as Halfabar,
is an aromatic densely-tufted grass growing wildly and widely in Upper Egypt.
The herb is highly reputed in folkloric medicine. In the present study, we describe
the mycoflora of the Cymbopogon schoenanthus L. Sprengel where thirty-eight
species in addition to 2 varieties belonging to 16 genera were isolated from the
plant leaves on glucose and cellulose-Czapek s agar at 28°C by using dilutionplate
method. The most frequently encountered fungal species on the two types of
media were: Aspergillus niger, A. terreus var. africanus, A. terreus var. aureus,,
Cochliobolus spicifer, Emericella nidulans , Fusarium dimerum and Penicillium
camembertii. Aqueous, methanol, ethyl acetate and n-butanol extracts of the plant
leaves were tested at different concentrations against 17 pathogenic and nonpathogenic
fungi. These extracts were also evaluated for their activity against some
pathogenic bacteria. Most of the studied microbes showed high sensitivity to all
tested fractions. Among the solvent extracts tested, methanol extracts gave more
effective than other solvent extracts. It will be necessary to identify the active
compounds or components and to evaluate their potential for use as antimicrobials.

Full text

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Original Research Article
Mycoflora associated with
Halfa
-
bar
leaves and stems (
Cymbopogon
schoenanthus
L
.
Sprengel)
,
in vitro the antimicrobial activity of the plant
leaves and stems secondary metabolites
A.
Sabry
1,
S.A.El
-
Zayat
2
, A. H. M. El
-
Sai
d1
, F.F. Abdel
-
Motaal
2,
and
T.A. Magraby
1
1
Botany Department, Faculty of Science, South Valley University, Qena, Egypt
2
Botany Department, Faculty of Science, South Valley University, Aswan Egypt
*
Corresponding author
A B S T R A C T
Introduction
The genus
Cymbopogon
, a member of the
family Gramineae. It is a perennial herb,
erect, tufted 9cm. long, culms slender,
erect, glabrous 3-4 noded. Leaf simple,
alternate, linear 5-7 cm. long, sheathed
apex spiny entire.
Inf
lorescence spikelets
highly branched. 5cm long
(El tahir
et al
.,
2010).
It grows in
Southern
Egypt
and
Northern Parts of Sudan (Boulos, 1999).
Distributed in Central and Northern Sudan
(
Eltahir
et al., 2010). It is highly reputed
in Egyptian folk medicine as an effective
renal antispasmodic and diuretic agent.
(Taeckholm 1974 & Boulos 1983 &
Batanouny
et al., 1999;
El
-
Askary
et al.,
2003
).
The entire dried herb has been used
ISSN: 2319
-7706
Volume
3
Number
2
(201
4
) pp.
8
74
-882
http://
www.ijc
mas.com
Keyword s
Cymbopogon
schoenanthus
L.
; Sprengel;
mycoflora
;
antmicrobial
activity
;
secondary
metabolites.
Cymbopogon schoenanthus L
.
Sp
rengel, Family Poaceae, locally known as Halfa-
bar, is an aromatic densely
-
tufted grass growing wildly and widely in Upper Egypt.
The herb is highly reputed in folkloric medicine. In the present study, we describe
the mycoflora of the Cymbopogon schoenant
hus
L. Sprengel
where
thirty
-
eight
species in addition to 2 varieties belonging to 16 genera were isolated from the
plant leaves on glucose and cellulose-
Czapek
s agar at 28°C by using dilution-
plate method. The most frequently encountered fungal species on the two types of
media were: Aspergillus niger
,
A. terreus
var
. africanus
,
A. terreus
var.
aureus,
,
Cochliobolus spicifer
,
Emericella nidulans , Fusarium dimerum
and
Penicillium
camembertii.
Aqueous, methanol, ethyl acetate and n-butanol extracts of the plant
leaves were tested at different concentrations against 17 pathogenic and non-
pathogenic fungi. These extracts were also evaluated for their activity against some
pathogenic bacteria. Most of the studied microbes showed high sensitivity to all
tested fractions. Among the solvent extracts tested, methanol extracts gave more
effective than other solvent extracts. It will be necessary to identify the active
compounds or components and to evaluate their potential for use as antimi
crobials.

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for centuries by the Bisharin and Ababda
tribes of the Aswan Province in the form
of a decoction to produce diuresis, to
relieve colicky pains, to help the removal
of small stones from the urinary tracts, and
as an antipyretic in fevers. A bicyclic
sesquiterpene diol, proximadiol with
unique antispasmodic properties has been
isolated from
C.proximus
leaves (Radwan,
1975). Proximol® possesses unique
antispasmodic properties as it produces
relaxation of the smooth muscle fibers
without abolishing the propulsive
movement of the tissue (El-
Askary
et al.,
2003).
Materials and Methods
Tested Micro
-
organisms
The micro-organisms used in this study
consisted of: Five common fungal species
(A.flavus, A.niger, C.spicifer, F.dimerum,
M.circinelloides
)
as well as four crop
threatening pathogenic fungi, (
Alternaria
alternata
,
Cochliobolous spicifer
,
Stachybotrys atra var.
microspora
, and
Ulocladium botrytis) isolated from
Vicia
faba
. Fungal species were incubated on
Potato glucose agar (PGA) (Potato, 200 g;
Glucose, 20 g; Agar, 20 g and 1000 ml
distilled water) for 7 days at 28C
Eight
dermatophytic fungi (Candida albicans
,
Candida tropicalis
,
Candida
krusei
,Epidermophyton floccosum,
Trichophyton rubrum,
Trichophyton
mentagrophytes, Trichophyton verrucosum
and
Microsporium canis) collected from
As
suit fungal centre.
Dermatophytic fungi employed in the
screening on Sabouroud Glucose agar
medium at 28C. Sabouroud glucose agar
(SGA) is composed of Glucose, 40 g;
Peptone; 10 g; Cyclohexamide, 0.5 g;
Agar, 20 g per 1L distilled water for 15
days.
Three
pathogenic bacteria (
Staphylococcus
aureus
MARSA, Escherichia coli and
Salmonella
typhi
) collected from
bacteriology laboratory at Qena were
grown on nutrient agar medium at 37 C.
Nutrient agar composed of (peptone, 5g,
beef extract, 3g; agar, 15 g).
P
reparation of Plant Aqueous Extract
Powdered samples (100gm) of
Cymbopogom schoenanthus L. Sprengel
leaves and stems were macerated with
1000ml sterile distilled water in a blender
for 10 min. The macerate was first filtered
through double layered muslin cloth
followed by centrifugation at 4000rpm for
30 min. at room temperature. The
supernatant was filtered through
Whatmann No. 1 filter paper and
sterilized, which served as the mother
extract (Satish et al. 2007). For evaluation
of antifungal activity of the extracts,
percentage dilutions i.e. 15%, 20%, 30%
and 40% of extract were obtained by
adding appropriate of standard basic stock
solution to stock media.
Antimicrobial Activity Assay
For screening of antimicrobial activity of
powdered ingredients of C. schoenanthus
L. Sprengel poisoned food technique was
followed (Sinha et al. 1993). Potato
Dextrose Agar (PDA) medium was
prepared and sterilized. The medium was
supplemented with different serial
dilutions of aqueous extracts i.e. 15, 20,
30, & 40
% (stock solution). About 15ml of
this medium was poured into each
petriplate and allowed to solidify. Ten mm
disc of 7-
day
-old culture of each fungus
were placed at the centre of the each
petriplate and incubated at 28°C for 3, 4,
& 5 days . After incubation, the colony
diameter was measured in millimeter

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(mm). For each treatment group (for a
given percentage of extract) four replicates
were maintained. PDA medium without
the aqueous extract was taken as control
but in case of dermatophytes we used
Sabouro
ud Dextrose Agar (SDA) medium.
For pathogenic bacteria we used Nutrient
Agar (NA) medium. The fungitoxicity of
the extracts was taken in terms of
percentage inhibition of mycelial growth
was calculated by using the following
formula:
% inhibition
= dc
-
dt/dc x100
Where dc= Average increase in mycelial
growth in control, dt = Average increase in
mycelial growth in treatment (Singh and
Tripathi 1999).
Preparation of Organic Extract
Ten grams of dried plant material was
successively extracted with 100 ml of
methanol, 100 ml n-butanol and100 ml
ethylacetate kept on a rotary shaker for 24
h at room temperature (Al-
Fatimi
et al.
2007). The solvent concentrated under
vaccum, weighted and kept in frozen (-
20C) for antimicrobial assays.
Antimicrobial Activity Assay
Antimicrobial activity was studied using
filter paper disk diffusion method
(Benson, 1990). The degree of growth
inhibition was evaluated after 24hr for
bacteria and 48hr for fungi and compared
with the growth inhibition results obtained
from the controls
.
Results and Discussion
Mycoflora associated with Cymbopogon
schoenanthus
L.Sprengel
The dilution plate method showed that
C.
schoenanthus
L. Sprengel leaves and
stems were highly contaminated with
various types of fungi
where
thirty
-e
ight
species in addition to 2 varieties belonging
to 16 genera were isolated from the plant
leaves and stems on glucose and cellulose-
Czapek
's agar at 28°C. The most
frequently encountered fungal species on
the two types of media were:
Aspergillus
niger,
A. terreus
var
. africanus, A. terreus
var.
aureus,,
Cochliobolus spicifer,
Emericella nidulans , Fusarium dimerum
and
Penicillium camembertii. Data shown
in Table (1). The results agreed with that
obtained by Abou Donia, 2008. That
found
Aspergillus, Fusarium
and
Penicillium
genera were more frequently
detected than other genera of fungi on
spices and medicinal plants samples.
Aspergillus
spp. was found in all examined
medicinal plant samples under
investigation. Bungo et al., 2006. reported
that the predominant mycoflora on ninety-
one samples of medicinal plants was
distributed in 10 genera. From these
89.9% of the isolates corresponded to
genera
Aspergillus and Penicillium.
Moharram
et al. (1989) and Regina and
Raman, (1993) reported that the fungi of
Aspe
rgillus
spp.,
Penicillium
spp,
Rhizopus
spp. and
Fusarium
spp. are
contaminated (anise, cumin, coriander,
carawy and fennel) which the most
important medicinal and aromatic seeds in
Egypt and in the world.
Antimicrobial Activity
The aqueous extract of C.
schoenanthus
L.
Sprengel
showed antimicrobial activity
against the tested fungi and bacteria while
F.dimerum, U.botrytis, C.albicans,
C.tropicalis, E.floccosum and
M.canis
were tolerant to Halfa-bar aqueous
extracts. Results showed in figure (1). The
org
anic extracts (Methanol, Ethylacetate
and N
-
butanol) were more effective
than

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Table.1
Average
total counts , maximum values(calculated per g dry leaves and stems in all samples), number of cases of
isolation (NCI, out of 20 samples) and occurrence re
marks (OR) of fungal genera and species recovered from 20 samples of
Cymbopogon schoenanthus
L. Sprengel
leaves and stems on glucose
-
and cellulose
-Czapek sagar at 28 oC
Cellulose
Glucose
NCI&OR
ATC±SD (MV)
NCI&OR
ATC±SD (MV)
Genera an
d specie
2 R
50 ±0.57735 (1)
3 L
125 ± 1.892969 (4)
Acremonium
1 R
25000± 0.5 (1)
A.fusidiodes
4 L
125000± 1.258306 (3)
A.kiliense
2 R
50000 ±0.57
735 (1)
A.rutilum
1 R
125000 ±1.5 (3)
2 R
100000± 2 (4)
A.strictum
1 R
25000± 0.5 (1)
Alternaria
1 R
25000± 0.5 (1)
A.alternata
18 H
11650000±14.34108 (135)
19 H
14425000 ±19.20069 (160)
Aspergillus
1 R
25000± 0
.5 (1)
A.carenus
3 L
100000± 0 (1)
3 L
150000± 1.290994 (3)
A.flavus
3 L
1100000± 4.320494 (15)
3 L
1425000± 7.274384 (21)
A.fumigatus
1 R
25000± 0.5 (1)
A.granulosus
14 H
3025000± 5.439056 (38)
15 H
3700000± 6.055301 (41)
A.niger
2 R
50000±0.57735 (1)
2 R
75000± 0.957427 (2)
A.sydowii
7 M
1725000±2.872281 (19)
17 H
6750000± 9.848858 (80)
A.terreus
var
. africanus
14 H
5650000± 9.146948 (63)
10 H
2275000± 5.619905 (29)
A.terreus
var
. aureus
14 H
750000 ±7.
767453 (19)
13 H
775000 ±2.5 (11)
Cochliobolus
2 R
75000± 0.957427 (2)
C.lunatus
12 H
675000± 7.632169 (18)
13 H
775000 ±2.5 (11)
C.spicifer
1 R
25000± 0.5 (1)
Coleophoma cylindrospora
1 R
25000± 0.5 (1)
Cladosporium
1 R
25000± 0
.5 (1)
C.sphaerospermum
3 L
75000± 0.95742 (2)
2 R
50000 ±1 (2)
Curvularia
2 R
50000± 0.57735 (1)
2 R
50000 ±1 (2)
C.ovoidea
1 R
25000 ±0.5 (1)
C.pallescens

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Table (1) : Cont.
Cellulose
Glucose
NCI&OR
ATC±SD (MV)
NCI&OR
ATC±SD (MV)
Genera and specie
s
9 M
525000± 4.787136 (12)
6 M
475000 ±1.5 (6)
Emericella
9 M
525000± 4.787136 (12)
6 M
475000 ±1.5 (6)
E.nidulans
8
M
1950000±6.027714 (28)
12 H
1050000± 7.593857 (21)
Fusarium
8 M
1950000±6.027714 (28)
12 H
1050000± 7.593857 (21)
F.dimerum
3 L
75000± 0.5 (1)
2 R
75000± 0.957427 (2)
Gibberella
2 R
50000± 0.57735 (1)
1 R
50000± 0.57735 (1)
G.fujikuroi
1 R
25000± 0.5 (1)
1 R
25000 ±0.5 (1)
G.tricincta
1 R
25000± 0.5 (1)
Hypocrea
1 R
25000± 0.5 (1)
H.rufa
1 R
25000 ±0.5 (1)
Mucor
1 R
25000 ±0.5 (1)
M.circinelloides
11 H
145000± 4.99166 (22)
13 H
850000 ±3.696846 (14)
Penic
illium
4 L
525000± 2.886751 (8)
8 M
450000± 1.290994 (6)
P.camembertii
1 R
25000± 0.5 (1)
P.chrysogenum
1 R
75000± 0.957427 (2)
P.citrinum
4 L
125000 ±1.290994 (3)
1 R
25000± 0.5 (1)
P.corylophilum
1 R
50000± 1 (2)
P.dendrit
icum
4 L
325000± 0.5 (6)
P.duclauxii
1 R
25000± 0.5 (1)
2 R
75000± 1.5 (3)
P.funiclosum
1 R
25000± 0.5 (1)
2 R
100000 ±1.414214 (3)
P.oxalicum
1 R
75000± 1.5 (3)
1 R
25000 ±0.5 (1)
P.puberulum
1 R
25000± 0.5 (1)
P.viridicatum
1 R
300000± 1.154701 (4)
P.waksmani
6 M
375000± 2.380476 (5)
8 M
475000± 1.258306 (6)
Phoma
1 R
50000 ±0.57735 (1)
P.eupyrena
3 L
175000±1 (2)
2 R
75000± 0.957427 (2)
P.exigua
3 L
150000 ±1 (2)
8 M
400000± 1.825742 (6)
P.medicag
inis

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Table (1): Cont
.
1 R
25000± 0.5 (1)
Scytalidium
1 R
25000± 0.5 (1)
S.lignicola
5 M
125000 ± 0.5 (2
7 M
325000± 2.061553 (5)
Sterile mycelia(white & dark colour)
17225000±11.34313
18825000±14.77329
Gross total count
13 13
Number of genera
27+2var.
29+2var.
Number of species
Fig.
1 Antimicrobial activity of Cymbopogon schoenanthus L. Sprengel aqueous extract on the growth of tested fungi and bacteria at
different concentrations
.

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Fig.2
Antimicrobial activity of
C. schoenanthus
L. Sprengel organic extract
on the growth of tested fungi and bacteria

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the aqueous extract where it showed high
antifungal activity against the tested fungi
but
A.flavus, F.dimerum, S.atra
var.microspora, C.albicans, C.tropicalis,
C.krusei, E.floccosum, M.canis, T.rubrum
and T.verrucosum
were tolerant to these
fractions. Organic extraction of Halfa-
bar
showed high antibacterial activity against
all the tested pathogenic bacteria (
S.aureus
MARSA,
S.typhi
and
E.coli
).
Results showed in figures (2). The present
results are inconformity with other studies,
where halfa bar was shown to be the most
potent among many tested plant extracts
against
Fusarium verticillioides
and
Aspergillus flavus (El-
Assuity
et al.,
2006). Proximadiol, 5 -
hydroxy
- -
eudesmol, 1 -
hydroxy
- -eudesmol, 1 -
hydroxy
- -eudesmol, 5 -
hydroperoxy
- -
eudesmol and 7 , 11-
dihydroxycadin
-
10(14)
-ene were isolated from the
unsaponifiable fraction of extract of
Cymbopogon proximus. These
components were shown to be powerful
antimicrobial agents (El-
Askary
et al.,
2003). Fawzi et al., 2009 found that halfa
bar was the most efficient among many
tested plant extracts against
Alternaria
alternata
and Fusarium oxysporum and it
might be a promising material to c
ontrol
the studied fungi.
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